text:
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text:
Perennial Irrigated Pastures. III. Beef Calf Production from Irrigated Pasture and Winter Annual Range1
- C. A. Raguse,
- J. L. Hull and
- R. E. Delmas2
Irrigated pastures in the Western arid and semi-arid regions provide support to the beef cattle industry through the maintenance of beef cow herds and the production of calves. Increasingly unfavorable production economics, including the rising cost and logistical difficulty of seasonally moving cattle from seasonal range to other pastures (e.g., high mountain Federal granng allotments), necessitate evaluation of management options which permit increased stocking rate and year-around home ranch operation.
The objective of this study was to compare simple (range-only, yearlong) and complex [improved range and irrigated pasture plus winter supplementation with alfalfa (Medicago sativa L.) cubes] systems for beef calf production using a fall calving (November-December), early summer weaning (May-June) reproduction cycle.
In the complex system, cleared and reseeded range was used (winter) an average (4 years) of 6 months per year at a stocking rate of 2.2 ha/cow (2.8 AUM/ha). Irrigated pasture was used (summer) an average of 6 months per year at a stocking rate of 0.5 ha/cow (10.3 AUM/ha). Alfalfa cube supplement was fed (about 1 kg/ha per day) during fall and winter. In the simple system, cleared range was used yearlong at an average stocking rate of 5 ha/cow (2.4 AUM/ha). No supplements were fed except during the last year when drought prevailed. An average of 70 kg calf/ha was produced in the complex system. Average birth weight was 34 kg and average Lily gain (ADG) to 205 days was 0.76 kg. In the simple system, comparable values were 37.5 kg calf/ha, 32 kg birth weight, and 0.75 ADG. Birth weights and ADGs were not significantly different between systems. Average cow weights reflected seasonal changes in forage availability and quality. Cows in the simple system tended to lose weight more consistently during the months July. October than did those in the complex system, which, instead, more consistently lost weight during the months November-March. Economic return was similar but low in both systems. While total land per cow/calf unit was less for the complex system and total investment higher for the simple system, operating costs, depreciation, and interest were higher for the complex system.
It appeared that second and third trimester cow stresses, as evidenced by negative bodyweight changes, did not adversely affect calf birth weights and ADG, at least at the levels found under these experimental conditions.Please view the pdf by using the Full Text (PDF) link under 'View' to the left.
Copyright © . .


text: The ultimate shopping spree is just minutes from your hotel doorstep along side the many bars restaurant and attractions. Ideally located for a weekend away the hotel offers a range of facilities such as a leisure club sauna and steam room other additional services are at a supplement. The hotel offers everything to cater for your needs after a busy day.


text:
Venezuela's Maduro cuts prices of toys, computers in 'Merry Christmas' plan
By Corina Pons
CARACAS Nov 13 (Reuters) - In Venezuela, Christmas has begun in November.
President Nicolas Maduro has launched a plan dubbed "Operation Merry Christmas" to lower the cost of toys, clothes and appliances for shoppers, potentially boosting his slumping poll numbers as the country struggles with a weakening economy.
With soldiers selling subsidized holiday food and some 27,000 government inspectors leaning on private businesses to cut prices, Maduro hopes to take the edge off the country's 63 percent inflation during the Christmas shopping season.
That may help him improve approval ratings that dropped to 30 percent in September, according to pollster Datanalisis.
The government's price control czar Andres Mendez kicked off the holiday shopping season earlier this month with orders that businesses keep prices down and warnings that merchants must not charge more than 30 percent markup on any item.
Within days, shops were slashing prices and soldiers were organizing Christmas 'fairs' full of subsidized goods from food to electronics.
One throng of people gathered outside a downtown Caracas toy store to pick up cut-rate Barbie dolls that had been marked down by nearly 80 percent to 553 bolivars, only around $5 at the black market exchange rate.
"I'm buying six Barbies before they run out," said Carmen Suarez, a mother of two who got permission to leave work. "They said I could buy six as long as I didn't buy the same model twice." Continuación...


text:
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text:
On board MS BREMEN from Longyearbyen to Kangerlussuaq
Expedition to Spitsbergen, Iceland and Greenland - A trio of explorations on Arctic coasts
Which facet of the Arctic is the most spectacular, the most beautiful? Find out on a thrilling expedition to extraordinary highlights in the expanse of the North Sea. Spitsbergen, Iceland and Greenland with its seldom-visited east coast: three worlds, one adventure. Here, the BREMEN will take full advantage of its ice class and Zodiac rides will enable you to enjoy intensive nature and wildlife sightings.
from Longyearbyen to Kangerlussuaq
|Cruise dates:
|from 05.07.2016 to 22.07.2016 (17 days)
|Your ship:
|Cruise number:
| Tue, 05.07.2016
Charter flight Duesseldorf** – Longyearbyen
| Wed, 06.07.2016
- Sat, 09.07.2016
Spitsbergen (including Moffen, Liefdefjord, Amsterdamoa, Magdalenefjord, Ny-Alesund, Moellerhafen (Cross Bay))
Nordic natural spectacles in Spitsbergen
Look forward to visiting "Norway's cold coast": the island group – with the peaked mountains that inspired its name, mighty glaciers, deep fjords and untouched wilderness – is the perfect terrain for passionate explorers. With the highest ice class for passenger ships and a crew with years of Arctic experience, the BREMEN allows you to enjoy particularly intensive expedition moments. The Zodiacs will take you right to the heart of the adventure when the weather and ice permit you to explore the natural surroundings. With a little luck, polar bears, whales, Arctic foxes and seals will appear in this world of ice and glaciers for intensive wildlife encounters. For example off the small island of Moffen, famous for the large walrus herds that gather on the flat shingle beach. Perhaps you will spot some polar bears on the coast in the Liefdefjord while you marvel at the blue, shimmering ridge of the mighty Monaco Glacier. The uninhabited island of Amsterdamoa offers an almost surreal panorama. Several small lakes, formed due to the glacier, contrast with the glistening ice. Cast your gaze over majestic mountain landscapes in the Magdalenefjord, and discover traces of the history of whaling. Ny-Alesund is now an international centre for Arctic research. Numerous seabirds call this area home, alongside a few scientists focusing on climate change. Visit the world's most northerly "bar" in Moellerhafen – and be surprised.
| Sun, 10.07.2016
Relaxation at sea
| Mon, 11.07.2016
Cruising off Jan Mayen
The silhouette of the mountains in the archipelago, which rise up to 1,700 m, remain visible as the BREMEN heads for the Arctic Ocean – until an active volcano gets you in the mood for the power and magic of Iceland off the inhospitable island of Jan Mayen.
| Tue, 12.07.2016
Relaxation at sea
| Wed, 13.07.2016
Reykjavik/Iceland, 8.00 – 19.00 hrs
Iceland: the queen of the elements
The impressive forces of nature beneath Iceland's soil are reflected in the name of the capital, Reykjavik, or "Smoky Bay". The largest volcanic island on the planet is a wonderland all of its own. Gain spectacular insights with the Gullfoss, geysers and Thingvellir – the Golden Circle* – or marvel at breathtaking scenes on an aerial tour above the island of fire and ice* before the BREMEN sets sail for Greenland.
| Thu, 14.07.2016
Relaxation at sea
| Fri, 15.07.2016
- Sat, 16.07.2016
Eastern coast of Greenland
Overwhelming natural beauty in the world of the Inuit
A pure expedition feeling will accompany you throughout your exploration of Greenland. The BREMEN will head to the seldom-visited east coast, where the unpredictable ice conditions will determine what happens. Raw, untouched, barely inhabited – with its rugged mountain and fjord landscape, Ammassalik is a hiker's paradise. Here, as on the entire route, you will have many good opportunities for whale watching. This will also be the case in the Skjoldungen Sound, surrounded by glaciers, where you will get into the Zodiacs and embark on a hike in the lush tundra of southern Greenland.
| Sun, 17.07.2016
Passage through Prins Christian Sund (depending on ice)
With turquoise water, imposing glaciers and waterfalls, Prins Christian Sund presents an exhilarating, monumental natural scene.
| Mon, 18.07.2016
- Thu, 21.07.2016
Western coast of Greenland
The old colonial buildings in the harbour of Paamiut are very picturesque. The south-western coast of Greenland is famous for its large population of humpback and fin whales at this time of year. Take your front-row seats for the next spectacular nature experience: from the deck of the BREMEN, cruise the Sermilinguaqfjord to watch nesting puffins, razorbills and black guillemots in the rocks. Filled with tradition and history, the Inuit culture is not just reflected in the whalebone gateway at Qeqertarsuaq. This town on Disko Island was once a flourishing whalers' settlement and is surrounded by fascinating hot springs and rugged basalt cliffs. And then you will see it in front of you – Disko Bay. Countless icebergs slowly drift through the bay, while the Arctic sun bathes the landscape in a magical light – and you will cruise and ride right through it on the BREMEN and in the Zodiacs, respectively. With contrasts such as the traditional and modern, whaling history and nature, the small town of Sisimiut perfectly embodies your Arctic explorer's dream.
| Fri, 22.07.2016
Kangerlussuaq/Greenland, arrival 6.00 hrs
* The shore activities are not included in the cruise price and are in the planning stage. Subject to changes.
** Depending on the flight schedule, it may be necessary to change the airports of departure and arrival.
A change in itinerary may become necessary on this expedition. The captain will decide on the best possible alternative depending on weather and ice conditions.
= at anchor
Please note that our pre- and post-cruise programmes are conducted in German.
For individual programmes kindly consult your travel agent.
Following programmes we offer in German:
A geology specialist in the team of experts
Dr Ulrich Dornsiepen, who has led numerous scientific excursions, projects and research groups, presents his knowledge in a vivid manner. This university professor of geology will present fascinating talks on subjects such as geodynamics.


text:
- 2.4"w x 3.2"h
- 10 Total Watts
- 1 Light
- Xenon Output
Delicate transparent cone-shaped glass shade with fluted bottom and decorative glass spiral topped with glass ball detail. Includes (1) 20 watt low-voltage G4 base Xenon bi-pin lamp and 6' of field-cuttable suspension cable. For use with 24 volt transformer, add suffix "-24" to item number.
The LBL HS447CRSC1B10MPT Bling collection Pendant Lighting in Satin Nickel uses Xenon light output and brings Modern style. This item is 3.2"H x 2.4"W. The total wattage is 10 and the voltage is 12 volts. The LBL HS447CRSC1B10MPT requires 1 bulb (included). Additional Information: Shade-Sandblasted Crystal; Accessory-4" Round Flush Canopy in Satin Nickel.


text:
Cleveland Clinic Foundation, Cleveland, OH
PURPOSE: Literature addressing the acute effects of sleep apnea in patients undergoing coronary artery bypass grafting (CABG) and other cardiac surgeries, which are commonly performed in patients with CAD, is sparse. The objective of this retrospective study is to compare the incidence of perioperative complications in patients with and without OSA who undergo cardiac surgery.
METHODS: Inclusion criteria: The Cleveland Clinic Cardiac Surgery and Sleep Center databases were used to identify the patients who underwent an open heart surgery within two years of the date of the polysomnogram (PSG) between January 1995 and December 2000. An assumption was made that if the surgery was performed within two years of the diagnosis of OSA, the patient had OSA at the time of the surgery. During this period 25,587 patients were identified in the cardiac surgery database of which 37 were diagnosed with OSA by PSG.To balance the differences and reduce bias propensity analysis was used to select a sample from patients without OSA.
RESULTS: Higher incidence of encephalopathy (p=0.008), postoperative infection (0.028) and increased ICU length of stay (p=0.031) were noted in the group with OSA after cardiac surgery. The difference in the rates of infection was mostly accounted for by the presence of mediastinitis (8.1% vs 1.08%). This could be attributed to prolonged ventilator support and higher length of ICU stay. However,differences in the rates of reintubation, tube time, and overall postoperative morbidity were not statistically significant.
CONCLUSION: Increased risk for postoperative complications is suggested in patients with OSA undergoing cardiac surgery. This risk is underestimated on account of lack of awareness about the incidence of OSA in the general population and the cardiovascular population in particular, difficulties in clinical suspicion and diagnosis and limited use of polysomnography.
CLINICAL IMPLICATIONS: OSA may need to be identified and diagnosed prior to cardiovascular surgery. Patients with OSA are more often diabetic but have an independently higher tendency to develop infections viz., mediastinitis.They also had higher rates of encephalopathy and longer ICU stay postoperatively.
DISCLOSURE: Roop Kaw, None.
Wednesday, October 25, 2006
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text:
Bacon Mushroom Egg Muffins for Office LunchMonday, January 20th, 2014
Long weekends always have me dreaming of all the extra stuff I'll get done on that extra day off. Rarely does reality actually meet my expectations! Today I am pleased to say however that I spent the entire day in my pajamas rearranging kitchen cabinets and catching up on laundry. You see for me staying in my pajamas is a rare occurrence that I don't often get to enjoy! Bet you thought I was pleased about the cabinets and laundry lol? Well maybe that too!
So with all my "extra" time I decided to make a batch of gluten-free, bacon-mushroom egg muffins. I had leftover mushrooms (purchased for homemade beef stew) and I had leftover bacon (purchased for bacon cabbage stir-fry) so that determined the kind of egg muffins that I would make. I call these egg muffins as opposed to frittata or quiche since I don't make a crust nor do I add flour to my egg mixture. That makes these easily gluten-free. I must confess when I am cooking for my family I often don't measure my ingredients. I am the queen of eyeballing and adjusting recipes to fit what I have or what i think my family will like better. While this does categorize me as a heck of a home cook, it does make me a poor food blogger ! Good thing I am not in it for the money!
So here are the general unmeasured instructions for bacon-mushroom egg muffins…
In your frying pan cook up onions, mushroom and sliced bacon for your egg muffin filling (spinach would be an excellent addition if I had it on hand). The more muffins you plan on making, the more filling mixture you should make. Divide your cooked filling by placing a couple spoonfuls in each cup of your muffin tin (spray muffin tin with cooking spray first). Top each muffin cup with shredded sharp white cheddar cheese (or your favorite cheese). In a bowl beat your eggs with milk. As a general rule of thumb you will need one egg for each muffin you plan to make. Season with salt, pepper, garlic powder and parsley. Pour the egg mixture over each muffin cup until filled to the top. Bake your muffins at 350 degrees until the egg muffins rise and are firm to the touch. They will sink upon cooling.
Served in an Easylunchboxes container, this lunch contains:
- 3 bacon-mushroom egg muffins (pack when cooled completely)
- 1 strawberry
- Sliced Cara Cara Oranges
- Gluten-free pretzel sticks and Snap Pea Crisps
Want one? (Note these links and others contained in this post are affiliate links)


text:
Pouring rain like a nonstop crescendo
with lightning and angry thunder
Birds gone home early, hungry too.
braving a long and stormy night
sleeping under raging skies.
Will the grey, stray cat called Jade
find his way back home before dark?
Over heavily falling rain it remains
a turbulent and an anxious wait.
PS. 1. The photos are by the courtesy of Google, so they are not mine!
2. My words describe last night!


text:
Henry H001T .22 S,L,LR
Guns International #: 100821363 Seller's Inventory #: 4897629
Category: Henry Rifles - Rimfire American Rifles
Seller's Information
When emailing or calling sellers direct, please mention that you saw their listing on GunsInternational.com
Seller: Cabelas East Hartford
Company: Cabela's
Member Since: 11/1/07
First Name: Joe
Last Name: Norton
State: Connecticut
Country: United States
Phone: (860) 290-6210
Fax: (860) 290-6242
Seller: FFL Dealer
Return Policy: 3 day inspection and return policy on used guns.
3 days
Payment Types Accepted:
Price: $599.99
Caliber: .22 S, L, LR
Metal Condition: Very good but with scratching to the underside of the reciver down to the metal(see picture)
Wood Condition: Excellent, with very minimal signs of wear
Bore Condition: Bright and shiny
Barrels: 20
Stock: Stright smooth stock
Fore End: LOP: 13.75
Butt Pad: Black plastic plate.
Weight: 6.75 lb
Sights: Front blade with a Konus pro 2-7X32 Scope and the rear sight has been removed.
Item Location: East Hartford, CT


text:
Gorgeous sunsets on Arbutus Lake are awaiting you on this well maintained year round 3 bedroom, 1 bath home. It features a roomy living room area and 3 decent sized bedrooms along with a 2 car attached garage, with a screened in area for more living space. The frontage is child friendly which is level to the lake. Call today!!!
Features Residential
|Lake Chain: 0
|Garage: 2 Car, Attached
|Lake Name: Arbutus Lake
|Year Built: 1970
|River Name:
|Basement Type:
|Water Frontage (feet): 0
|Fin SqFt Total: 1,087
|Lake Size-Acres: 161
|Lot Size: 55321
|High School:
|Site Influence: Casino Nearby, Hunting, Lake Front, Level, No Thru Road, Recreation Nearby
|School District:
|Construction: Frame/Stick, Year Round


text:
FS:2007 Wrangler Rubicon 2 Door
4 Attachment(s)
2007 Rubicon 2 door
Power Windows
Power Locks
6 Speed Manual
2" lift
33'' Pro Comp Extreme MT's on 17" Dick Cepeks Matching Spare
Rancho RS9000X adjustable shocks
Hard Top with (Black factory color)
Silver doors in excellent shape
Bikini Top
In excellent mechanical condition!
I'll take $17,900
Good luck with the sale bud, great price.
Can you post the milage? It will save a lot of questions for it. ;)
I knew I forgot something. I've never off roaded it, tires are about 70% good, the spare has never been used. It has 86,000ish on it. I still drive it so final mileage will be a little higher. Keep in mind it is a used vehicle, it's not perfect but it's deff not rough! Overall I rate it at an 8. I would have no problem driving this Jeep across the country! Please ask questions if you have them, no negative feedback please, I get enough of that from CL!
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text:
Initiated in 2012 by the United Nations, it acknowledges that happiness is a fundamental human goal and calls upon countries to help improve the well-being of all peoples. But imagine if it acknowledged that all beings desire happiness – which of course they do – and urged countries to improve the well-being of non-human beings also. Obviously one cannot bring happiness into the lives of individuals by imprisoning them and killing them, so farms and slaughterhouses would need to be phased out along with animal research facilities. All entertainment that uses and abuses animals would go too, including hunting and fishing.
"May all sentient beings be happy and free of suffering," said Gautama Buddha.
We may not be able to prevent all suffering but we can certainly eliminate a lot of it by our actions. If we seek happiness for ourselves, isn't it only fair that we allow others to be happy too?
Jenny Moxham
Victoria, Australia


text:
"What's sex?"
It wasn't so much that the question bothered her it was how she asked. The little girl had an air of high importance around her just like her father, much to Sakura's horror, and the glittery pink tiara proved it by showing the world she was the only Uchiha princess...for the time being of course.
She was going on six very soon and little Yuriko had an amazing ability to process and absorb information much quickly than other children her age. Sometimes Sasuke wished his daughter didn't take so much after her mother. It was annoying.
"Eh," Sakura looked over to Sasuke for help, a hand gently rubbing over her swollen tummy. She felt a strong kick and she smiled faintly imagining a little dark haired mama's boy…
"Mommy!" The girl stomped her foot impatiently effectively bringing Sakura out of a beautiful daydream where she was the one that was doted after by her children and husband.
Giving a defeated sigh she began to explain the concept of the birds and the bees, one of the most confusing and lamest example she had ever gotten as a young teenager from her parents. Kakashi's explanation of sex was much better (because he was blunt and not to mention he included pictures and charts), although she had been traumatized in the beginning for a while until it finally sunk in; she was more than thankful for the sex talk because a couple of years later she used that information to her advantage where it then led her to be Mrs. Uchiha Sasuke.
Sakura faintly heard Sasuke chuckling under his breath. The nerve of that man, dumping this on poor pregnant her! She huffed, hands going to her back for support, "Ask Daddy."
It was all it took for little Yuriko's charcoal eyes to glitter in pure happiness, squealing and running to Sasuke for an answer. Sakura was tempted to laugh evilly along with her inner self, but decided against it since it would ruin Sasuke's awkward father-daughter bonding.
- - - - - - - - - -
A/N: Another one-shot collection. This is actually from my lj community where I post picture challenges and the members have to write a fic using the drawing as inspiration. It's kinda slow right now, but whatever! I'll give the link away to anyone who is interested in joining.
community. livejournal. com/ tomatokiss / profile (delete spaces)


text:
Google Apps for Your Domain
November 27th, 2006 | Published in Google Analytics
Every now and then we will share updates from other Googlers about the products they're working on if we think that info will benefit Google Analytics users. Google Apps for Your Domain is similar to Google Analytics in that it offers simple and useful ways to view and share information online. And with these apps, you are actively creating information as well. Marlin Gilbert from the Google Apps team explains:
Like Google Analytics, Google Apps for Your Domain gives you the opportunity to focus your precious resources on campaigns and programs that rapidly grow your business. We built these tools to enable your team to communicate and collaborate more effectively without any additional hardware or software. Everything is online and accessible from any internet-connected device through a standard web browser. And you can customize the tools your company uses with your own special branding, color scheme and content.
Currently, you can take advantage of Gmail, Google Talk, Google Calendar and Google Page Creator and the new start page feature. For instance:
And we are working to make more apps available soon, so you can keep focusing on what you do best.
- Using Gmail, without changing your email addresses, your employees can enjoy a faster and clearer way to read email conversations, and find saved messages with powerful search tools.
- Coordinating complex schedules is a breeze with Google Calendar.
- Keep your people in touch through Google Talk and use Google Page Creator to make your web page ideas come to life.
- The start page is a central place for your employees to preview their inboxes and calendars, access your essential content, and search the web.
Google Apps for Your Domain is free, and currently in beta. Learn more and get started here.


text:
Sturm, Ruger & Company One Millionth Gun This Year
Massad Ayoob
Posted: 21 Aug 2012 07:19 AM PDT
With so much of the American economy foundering, it's no secret that one bright spot is the American firearms industry. Gun owners' fear of draconian legislation that may emanate from President Barack Obama, perennial top candidate for Gun Salesman of the Year, is unquestionably one factor driving this phenomenon.
Sturm, Ruger & Company has just made the following announcement:
Sturm, Ruger & Company, Inc. (NYSE: RGR) is on pace to beat its own record of 1,114,700 firearms produced in one year, set in 2011. On August 15, 2012 Ruger produced its one millionth firearm of the year, a Ruger® SR1911™ pistol which will be hand-engraved by Baron Technology, Inc. and auctioned off to support the National Rifle Association Institute for Legislative Action.
"Last year, Ruger became the first commercial firearms company to produce one million firearms in one year, and we were incredibly excited and proud to reach that milestone," said Ruger President and CEO Mike Fifer. "It took us nearly all of 2011 to build one million firearms, but in 2012 we accomplished it on August 15th. We continue to invest in and improve our manufacturing processes to help us respond to the strong demand for Ruger® firearms. We expect 2012 will be another record-breaking year for Ruger, and we want to thank our loyal customers for their continued support."
The firearms industry has seen consistent growth over the last few years, as measured by the National Instant Criminal Background Check System ("NICS") background checks as adjusted by the National Shooting Sports Foundation. In July 2012, NICS checks rose by 25.5% compared to July 2011, the 26th consecutive month that NICS checks have risen on a year-over-year basis. Demand for Ruger products has outpaced the growth in overall industry demand, driven by new product introductions including the Ruger American Rifle™, SR22™ pistol, SR1911™ pistol, and 10/22 Takedown™ rifle.
good for them
Seems their mindset is a bit better now that Bill Ruger is a ghost.I have a couple Rugers and have had my Ruger SBH run thru their shop.very good work.
i tried being reasonable,i didn't like it, NRA LIFE MEMBER,USMC VETERAN
My sis-in-law just brought her Ruger 10/22. She wanted me to
buy it. It does have a nice but dirty Simmons scope. I've never been a scope guy. The rifle has some rust, appears to have been last cleaned when the factory test fired it, yuck. There are a few minor scratches on the wooden stock.
My other .22s are a Remington Sportmaster that's almost the same age as me and a Monkey Wards single shot. Both good shooters :<))
sounds like
A 125 dollar gun.
The truth, the whole truth and nothing but the truth!
Thanks Vaq, kinda had $150 in mind but you're prolly closer
to the real value.
I figure the action to be the better part of a beat up 10/22. You can replace anything else with high end aftermarket parts. I've sold a few rifles with scopes attached. Never got one extra nickel for a scope.
The truth, the whole truth and nothing but the truth!
greg az
Daisy.. you got some sis-n-law there buddy,, evening guys..
Not knocking the gal Lew, we all have inlaws and outlaws.. but aint it grand when they want to charge ya tax.. Theres something about gals and guns, they always figure thier worth more than they really are.. had a gal up up Peeples Valley that was convinced her dads old police positive Colt was worth over 5 hundred bucks.. and (true story i checked the gun) it was so worn i thought it was nickle to start with.. nope had some blue left under the trigger guard, and the best part was it was locked up.. serious.. not out of time.. the whole cylinder was locked up to the point of not being able to turn it.. Colt but they made more police positives than any other revolver.. "might" have been worth 150.. Daisy you got mail..
whats this vaqueros on tonight.. you awake pard or sleeping from a hard day of that work you do.. BRAAAHHAAA..
"can i get you another cup of coffee sir".. MOOOOOOO
a man has to hold his word, hold his beliefs, and hold a good sight picture.
Yep, but Greg, that's not the 1/2 of it, did you get the note
about having Stella's ex-sis-in-law here for a week? Have mercy, I'm not coping very well but surviving.
Anyway, I always thought I'd enjoy a 10/22 but don't need one as much as I need a .22 pistole for the kids. That was the original conversation. Some way she got pistol and rifle confused :<)
The lady in PEOPLE'S Valley had what sounds like to me a "range gun" used in place of identical guns carried on duty when the cops used the range for practice or quals. Range guns could get bazillions of rounds through them saving the duty guns from extra wear.


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Undergraduate unit
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This Community Work Across Sites, Settings and Peoples unit gives students a thorough grounding in the diverse nature of community development within current Australian society.
Delivered online by Open Universities Australia in conjunction with Murdoch University, the unit aims to enhance students' abilities to analyse, interpret and critically reflect on community development campaigns by examining examples from real world contexts. These examples may include community development in the context of social work, urban planning, community services, remote area education, indigenous communities, overseas aid work, and sustainable development.
At the conclusion of this unit, students will have an in-depth understanding of community development practices and be able to analyse the principles they have studied in a variety of settings. Students will also become confident critically processing and applying information to a variety of community development situations.
Applicants must meet the criteria to be eligible to enter this program:
This unit is part of a major, minor, stream or specialisation in the following course:
Content may also be relevant to other courses, and could qualify you for Recognition of Prior Learning (RPL) for a course offered by another institution.
In this Community Work Across Sites, Settings and People unit, the topics you will study include:
This unit is geared towards students hoping to develop a career in community development work. You will be exposed to the fundamental background knowledge and practical skills currently used by teams working in community development and have the opportunity to apply these skills in a diverse range of situations: from local government and urban planning projects to overseas aid work and sustainable development programs.
By having a solid grasp of a range of highly sensitive and volatile topics such as civic rights and how to respond to suicide, you will be able to apply critical thinking skills when developing your own proposals. This will enable you to effectively function as a part of a team dedicated to improving local community services, infrastructure, atmosphere and relationships.
If you are an Australian citizen, you may be eligible to receive a government HECS-HELP or FEE-HELP loan, meaning you can defer payment of your fees.
This course can be paid for through the HECS-HELP or FEE-HELP government loan schemes. If you are an Australian citizen or hold a permanent humanitarian visa, this means you don't need to pay upfront.
Instead, the Australian government will pay your course fees on your behalf. You'll begin repaying your loan through the tax system once you start earning more than the minimum threshold of $54,869 (2016-17 income year).Find out more about government study loans here.
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Examine the way in which sustainability has evolved over the past four decades with this online Introduction to Sustainable Development unit from Murdoch University.
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GVI offers a range of long term volunteer opportunities in locations across the globe, which range from three months to one year. These options are ideal if you are looking to gain valuable practical experience in fields such as conservation or education, and many include extensive professional training opportunities.
Whether you're looking for a gap year, sabbatical, career break or a complete change in career direction, there's a project to suit you, including our internships abroad, expeditions and multi-country combos which offer a variety of long term volunteer programs.
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At Brave Luck Books all week through June 7th Savage Storm: Rys Rising Book II is on sale.
Save 30% off on the ebook (Kindle, PDF, or Epub available worldwide with no DRM).
No coupon codes. Just visit the Savage Storm page at Brave Luck Books and add to cart. Normally $4.95, fantasy fans can get the ebook this week for only $3.46.
This is no little novel. This is the second book of the Rys Rising series. Savage Storm is a 198,000-word novel. And if I do say so myself, it's one I'm rather proud of.
- You just have to read it. How else will you find out how Amar becomes the Overlord of the Kez?
- Nothing will take you away from your problems like feeling the terror of a grim winter siege at a frontier settlement overwhelmed by hordes of savages.
- Don't you want to witness the liberation of the rys city of Jingten as Dacian and Onja expel their cruel oppressors?
- Cities fall in this one and a Goddess is made.
- The thrones of the vanquished will be despoiled, I promise you!
Read more about the novel at my web store Brave Luck Books and save 30% this week on the ebook.
Book Excerpt from Savage Storm: Rys Rising Book II an epic fantasy of clashing civilizations. from Tracy Falbe


text: ATLANTA, May 14, 2013 /PRNewswire/ -- EarthLink, Inc. (NASDAQ: ELNK) today announced that it has caused ITC^DeltaCom, Inc., its wholly owned subsidiary, to commence a cash tender offer for any and all of the $292.3 million outstanding principal amount of ITC^DeltaCom's 10.5% Senior Secured Notes due 2016 (CUSIP No. 45031TAR5) (the "ITC^DeltaCom Notes"). In conjunction with the tender offer, ITC^DeltaCom is soliciting consents to eliminate most of the covenants, certain events of default applicable to the ITC^DeltaCom Notes and certain other provisions contained in the indenture governing the ITC^DeltaCom Notes and to release all of the collateral securing the ITC^DeltaCom Notes (the "Indenture"). EarthLink expects to fund the tender offer with the proceeds from a new senior secured debt offering. The tender offer is scheduled to expire at 11:59 p.m., New York City time, on June 11, 2013, unless extended or earlier terminated (the "Expiration Time"). Holders who validly tender their ITC^DeltaCom Notes and provide their consents to the amendments to the Indenture before 5:00 p.m., New York City time, on May 28, 2013, unless extended (the "Consent Expiration"), will be eligible to receive the Total Consideration (as defined below), which includes a consent payment. The tender offer contemplates an early settlement option, so that holders whose ITC^DeltaCom Notes are validly tendered prior to the Consent Expiration and accepted for purchase could receive payment of the Total Consideration as early as May 29, 2013. Holders that validly tender their ITC^DeltaCom Notes after the Consent Expiration will receive the Tender Offer Consideration (as defined below) promptly after the Expiration Time. Tenders of ITC^DeltaCom Notes may be validly withdrawn and consents may be validly revoked until the Withdrawal Time (as defined below). The "Total Consideration" for each $1,000 principal amount of ITC^DeltaCom Notes validly tendered and not validly withdrawn prior to the Consent Expiration is $1,058.75, which includes a consent payment of $30.00 per $1,000 principal amount of ITC^DeltaCom Notes. Holders tendering after the Consent Expiration will be eligible to receive only the "Tender Offer Consideration," which is $1,028.75 for each $1,000 principal amount of ITC^DeltaCom Notes. Holders will also receive accrued and unpaid interest from the last interest payment on the applicable ITC^DeltaCom Notes up to, but not including, the applicable settlement date for all of such ITC^DeltaCom Notes that we accept for purchase in the tender offer. Tendered ITC^DeltaCom Notes may be withdrawn and consents may be revoked before 5:00 p.m., New York City time, on the earlier to occur of (a) May 28, 2013 or (b) the date of execution of the Supplemental Indenture (as defined below), unless extended (the "Withdrawal Time"), but generally not afterwards. Any extension, termination or amendment of the tender offer will be followed as promptly as practicable by a public announcement thereof. The tender offer is subject to the satisfaction of certain conditions including: (1) receipt of consents to the amendments to the Indenture from holders of 66 2/3% of the principal amount of the outstanding ITC^DeltaCom Notes and execution of a supplemental indenture effecting such amendments (the "Supplemental Indenture"), (2) completion of the senior secured debt offering on satisfactory terms and (3) certain other customary conditions. ITC^DeltaCom may waive any of the foregoing conditions in its sole discretion.


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CIM file format description
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1 filename extension(s) found in our database.
.cim - Cimplicity HMI Screen
CIM file is a CIMPLICITY HMI Screen. CIMPLICITY HMI is a client/server based visualization and control solution that provides process visualization, data acquisition and supervisory control over manufacturing and process environments.
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Venturing slightly from their streetwear edge, Rick Owens rendered this blazer for a tailored appeal with a tan finish and slight cinch of the waist.
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La Fille Mal Gardee
The legendary Royal Ballet presents this pastoral ballet set to the music of Ferdinand Hérold, with choreography by Frederick Ashton. Prima ballerina Lesley Collier plays Lise, a young farm girl forced to marry against her heart's desire. John Lanchbery conducts this playful work featuring Garry Grant, Brian Shaw and Michael Coleman. This production of "La Fille Mal Gardée" was filmed for BBC television at Covent Garden in January 1981.
Rent DVDs for only $4.99 a month.
Movie Details
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- John Vernon
- Genres
- Special Interest, Ballet & Modern Dance, Dance
- Format
- Ratings
NRNot rated. This movie has not been rated by the MPAA.
Full Screen 1.33:1Subtitles
NoClosed captioned
NoLanguage and sound
English: Dolby Digital 2.0 StereoOther features
Color; interactive menus; scene access.
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Common Sense Note
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- Depends on your kid and your family
- Not appropriate for kids of the age most likely to want to see it


text: "Our lead product, PRT-201, represents a highly innovative approach to addressing the significant challenges associated with vascular access in patients with CKD, and we are very excited about the potential for PRT-201 to dramatically improve care for dialysis patients," said Timothy P. Noyes, President and CEO of Proteon. "Based on the positive results from this Phase 2 study, Proteon intends to advance PRT-201 into Phase 3 studies."Proteon is conducting a robust clinical research program evaluating PRT-201's ability to improve vascular outcomes. Proteon previously completed a Phase 1/2 AVF study, the results of which were recently published in the Journal of Vascular Access, and recently completed follow-up in a Phase 1/2 study in patients undergoing surgical placement of an arteriovenous graft (AVG). The AVG study results are expected in the first half of 2013. In October 2012, Proteon initiated enrollment in a Phase 1 study in which PRT-201 is delivered via a catheter following angioplasty of the superficial femoral or popliteal artery in patients with symptomatic peripheral artery disease. About PRT-201PRT-201 is an investigational recombinant human elastase that is being studied for its ability to improve outcomes in patients suffering from vascular disease. Elastase has been shown in certain preclinical settings to reduce neointimal hyperplasia formation and to cause dilation of segments of arteries and veins. These effects may decrease stenosis and prolong vessel patency. Improved patency may lead to fewer corrective surgical procedures, fewer hospitalizations, lower costs and less suffering for patients. PRT-201 has received fast track and orphan drug designations from the FDA for the indication of improving AVF and AVG surgical outcomes. About Proteon Therapeutics Proteon Therapeutics, Inc. is a privately held biopharmaceutical company developing novel, first-in-class pharmaceuticals to address the critical medical needs of patients with kidney and vascular diseases. The company is headquartered in Waltham, MA., and has research facilities in Kansas City, MO. For additional information, please visit www.proteontherapeutics.com . SOURCE Proteon Therapeutics, Inc.


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Tim23p is an essential channel-forming component of the multisubunit TIM23 complex of the mitochondrial inner membrane that mediates protein import. Radiolabeled Tim23p monocysteine mutants were imported in vitro, incorporated into functional TIM23 complexes, and subjected to chemical cross-linking. Three regions of proximity between Tim23p and other subunits of the TIM23 complex were identified: Tim17p and the first transmembrane segment of Tim23p; Tim50p and the C-terminal end of the Tim23p hydrophilic region; and the entire hydrophilic domains of Tim23p molecules. These regions of proximity reversibly change in response to changes in membrane potential across the inner membrane and also when a translocating substrate is trapped in the TIM23 complex. These structural changes reveal that the macromolecular arrangement within the TIM23 complex is dynamic and varies with the physiological state of the mitochondrion.
Mitochondria are morphologically complex organelles containing four subcompartments: the outer membrane (OM), the intermembrane space (IMS), the inner membrane (IM), and the matrix. Because most proteins that reside in mitochondria are nuclear-encoded and synthesized on cytosolic ribosomes, several multisubunit mitochondrial translocases are required to direct mitochondrial proteins to their correct locations (Bohnert et al., 2007 ; Neupert and Herrmann, 2007 ). Proteins targeted to mitochondria initially engage the TOM (translocase of the outer membrane) complex that mediates both translocation across, and integration into, the OM (Bohnert et al., 2007 ; Neupert and Herrmann, 2007 ). Two major translocases of the inner membrane (TIM) mediate the translocation and integration of specific sets of precursor proteins at the IM. The carrier translocase (TIM22 complex) mediates the integration of polytopic membrane proteins (MPs) into the IM in a manner that is strictly dependent on the membrane potential (Δψ) across the membrane (Rehling et al., 2004 ). The presequence translocase (TIM23 complex) mediates the Δψ-dependent translocation of soluble proteins with N-terminal presequences into the matrix, as well as the integration of single spanning MPs into the IM (Neupert and Brunner, 2002 ). Translocation of soluble substrates across the IM into the matrix requires the ATP-powered PAM (presequence translocase-associated motor) complex (Bohnert et al., 2007 ; Neupert and Herrmann, 2007 ).
The TIM23 complex of Saccharomyces cerevisiae consists of four integral membrane proteins: Tim23p, Tim17p, Tim50p, and Tim21p. Tim23p consists of a C-terminal membrane-bound region with four predicted transmembrane (TM) helices and a hydrophilic N-terminal region in the IMS. Electrophysiological studies have shown that Tim23p forms a voltage-gated, cation-selective channel (Lohret et al., 1997 ; Truscott et al., 2001 ); therefore Tim23p is thought to comprise at least part of the protein-conducting pathway through the IM. The multifunctional IMS region of Tim23p can be divided into two parts: the N-terminal half has been shown to span the OM and may help position the TIM23 complex relative to the OM (Donzeau et al., 2000 ), and the C-terminal half binds presequences and mediates the dimerization of Tim23p molecules through a heptad leucine repeat motif (Bauer et al., 1996 ). In addition, the Tim23p IMS region regulates channel activity (Truscott et al., 2001 ). The Δψ plays two major roles in Tim23p function: it regulates Tim23p dimerization (Bauer et al., 1996 ), and it provides an electrophoretic force necessary for pulling the basic presequence of matrix-targeted substrates across the IM (Neupert and Brunner, 2002 ). Tim17p is homologous to the C-terminal region of Tim23p, and although its function is less clear, recent studies indicate that it is involved in substrate-induced voltage gating of the TIM23 channel (Meier et al., 2005 ; Martinez-Caballero et al., 2007 ), and in recruitment of the PAM complex (Chacinska et al., 2005 ). Tim50p, which contains a single TM segment and a large C-terminal domain in the IMS region, is essential for the recognition of substrates emerging from the TOM complex and in their transfer to the TIM23 channel (Geissler et al., 2002 ; Yamamoto et al., 2002 ; Mokranjac et al., 2003 ) and has been implicated in maintaining the permeability barrier across the IM (Meinecke et al., 2006 ). Tim21p is a single-spanning IM protein with a C-terminal region that directly tethers the TIM23 and TOM complexes and is involved in the substrate-directed switching of the TIM23 complex between matrix translocation and inner membrane sorting modes (Chacinska et al., 2005 ; Mokranjac et al., 2005 ). The associated PAM complex, responsible for mediating the ATP-driven translocation of soluble substrates across the IM into the matrix, consists of mtHsp70 (matrix heat-shock protein), the nucleotide exchange factor Mge1, Pam16p/Tim16p, Pam18p/Tim14p, Pam17, and Tim44 (van der Laan et al., 2005 ; Neupert and Herrmann, 2007 ).
Several studies have addressed the quaternary structure of the TIM23 complex, particularly with respect to the essential membrane proteins Tim23p, Tim17p, and Tim50p. Tim23p and Tim17p appear to be present in the complex in equimolar amounts (Bömer et al., 1997 ; Ryan et al., 1998 ; Moro et al., 1999 ) and together comprise the core complex of the presequence translocase (Dekker et al., 1997 ). Tim23p monomers associate through their IMS regions (Bauer et al., 1996 ) and not through their hydrophobic regions (Ryan et al., 1998 ). However, the hydrophobic domain of Tim23p has been shown to physically interact with Tim17p (Ryan et al., 1998 ). In addition, the IMS domain of Tim50p interacts with the C-terminal part of the IMS region of Tim23p (Geissler et al., 2002 ; Yamamoto et al., 2002 ; Mokranjac et al., 2003 ).
This complex is also highly dynamic and versatile, showing marked changes in its activity and subunit composition under different conditions. For example, early work demonstrated that Tim23p existed in separate, dynamically interacting subcomplexes (Bömer et al., 1997 ) and that newly imported Tim subunits could be exchanged for endogenous subunits (Dekker et al., 1997 ). Recent work has shown that the subunit interactions of the TIM23 complex vary in response to polypeptide substrates. Transiting precursor proteins can stably connect the TIM23-PAM and TOM translocases to form a supercomplex (Chacinska et al., 2003 ; Frazier et al., 2003 ) and interaction with the TOM complex is mediated by Tim21p (Chacinska et al., 2005 ; Mokranjac et al., 2005 ). The TIM23 complex switches between different modular states depending on sorting information in the polypeptide substrate: an inner membrane sorting form of TIM23 associates with Tim21p but lacks the PAM complex, whereas a matrix import form of TIM23 associates with the PAM complex but loses its association with Tim21p (Chacinska et al., 2005 ).
To extend our understanding of TIM23 complex quaternary structure, regions in Tim23p adjacent to Tim17p, Tim50p, and other Tim23p molecules have been identified by chemical cross-linking. These regions of proximity reversibly change in response to changes in the Δψ and also to the presence of substrate. This high-resolution analysis refines our understanding of Tim23p subunit interactions and defines structural rearrangements within an assembled TIM23 complex.
The SP6-containing plasmid pJE29 (Ryan et al., 1998 ) was used to generate the library of Tim23p variants. The substitution of all endogenous Cys residues to Ala to create Tim23pΔCys, and the subsequent introduction of a Cys codon (5′-TGC-3′) at each selected location was done using the Quikchange protocol (Stratagene, La Jolla, CA). The primary sequence of each construct was confirmed by DNA sequencing. mRNA was transcribed in vitro using SP6 RNA polymerase and PCR-generated DNA fragments encoding the full-length Tim23p as described (Flanagan et al., 2003 ).
Mitochondria were isolated as described previously (Daum et al., 1982 ) from either wild-type strain D273-10B or from strain RJ474, a strain expressing Tim23p-HA (Tim23p with a triple hemagglutinin [HA] epitope at its carboxy terminus; Davis et al., 1998 ).
The recombinant protein pSu9-DHFR (consisting of the first 69 residues of subunit 9 of the FO-ATPase fused to full length dihydrofolate reductase with a C-terminal hexahistidine tag) was subcloned into the pET30a(+) vector (Novagen, Madison, WI) and expressed in BL21 Star (DE3) cells (Invitrogen, Carlsbad, CA). Variants of this protein containing single Cys sites in the presequence were created by site-directed mutagenesis as above. After growth to an A600 of ~0.8, cells were induced with 1 mM IPTG for 2.5 h, pelleted cells were disrupted by sonication, and protein was purified from cell lysate on a Ni-NTA matrix using an imidazole gradient (Stan et al., 2000 ).
Tim23p mRNA was translated (26°C, 40 min) in a wheat germ–based extract in the presence of [35S]Met (0.25 μCi/μl) as described (Flanagan et al., 2003 ). Mitochondrial import reactions (26°C, 20 min) were carried out in import buffer (0.25 M sucrose, 20 mM HEPES-KOH, pH 7.5, 80 mM KCl, 5 mM MgCl2, 2 mM potassium phosphate, pH 7.5, 3 mg/ml bovine serum albumin [BSA; fatty acid-free], 3 mM NADH, 1.5 mM ATP) containing mitochondria (0.25 mg/ml) and translation reaction (8% vol/vol). After import, mitochondria were sedimented (12,500 × g, 5 min) and resuspended in cross-linking buffer (CB, Import buffer lacking BSA). For dissipation of the IM potential after Tim23p import, ionophores and uncouplers were added as indicated from ethanol stocks (final ethanol concentration = 0.06% vol/vol). Where indicated, purified pSu9-DHFR was added in the indicated concentrations in the presence of 2 μM methotrexate (MTX). Osmotic shock to disrupt the OM was performed as before (Davis et al., 1998 ). For proteolysis experiments, samples were treated with 50 μg/ml proteinase K for 20 min on ice followed by the addition of 1 mM phenylmethylsulfonyl fluoride (PMSF).
For chemical cross-linking, the homobifunctional thiol reactive reagent Bis-maleimidoethane (BMOE) was added from a dimethyl sulfoxide (DMSO) stock to a final concentration of 50 μM (final DMSO concentration = 2.5% vol/vol) to mitochondria (0.13 mg/ml) in CB. The pH of the incubation was maintained within the proper range to ensure that the maleimide group will react specifically with sulfhydryls (Smyth et al., 1964 ). For cysteine labeling, the reagent 4-acetamido-4′-[(iodoacetyl)amino] stilbene-2,2′-disulfonic acid (IASD) was added to a final concentration of 4.5 mM to mitochondria as above in CB with 1 mM dithiothreitol (DTT). BMOE cross-linking and IASD labeling reactions were incubated at 26°C for 20 and 10 min, respectively, and quenched by the addition of 40 mM DTT (cross-linking reactions) or 200 mM DTT (labeling reactions).
For immunoprecipitation after cross-linking reactions, mitochondria (25 μg) were sedimented and the pellets were resuspended in 100 μl of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% (vol/vol) SDS. Solubilized mitochondria were diluted 10-fold with lysis buffer (10 mM Tris-HCl, pH 7.5, 10–150 mM NaCl, depending on antiserum used, 5 mM EDTA, 1 mM PMSF, and 1% Triton X-100, vol/vol), and a clarifying spin (18,000 × g, 10 min) was used to remove undissolved material. Supernatants were transferred to new tubes, and protein A-Sepharose beads precoupled with antibody were added. After overnight rocking at 4°C, beads were washed three times in 1 ml of the respective lysis buffer, once in 1 ml of 10 mM Tris-HCl (pH 7.5), and eluted with 30 μl of SDS-sample buffer.
For coimmunoprecipitation, mitochondria (125 μg) were sedimented and solubilized in 250 μl ice-cold solubilization buffer (30 mM HEPES-KOH [pH 7.5],10 mM NaCl, 0.5 mM EDTA, 10% [vol/vol] glycerol, 1% [vol/vol] digitonin [2× recrystallized in ethanol]), 1 mM PMSF, and standard protease inhibitors]. After incubation (4°C, 30 min), the extract was cleared by centrifugation (12,500 × g for 10 min), the supernatant was rocked with 8 μl of respective antibody (4°C, 2.5 h), and incubation with precoupled beads and elution was performed as above.
The objective of this work was to identify TIM23 complex subunits that are proximal to specific regions of Tim23p and to determine how their proximity may vary with changes in membrane potential and with the presence of substrate. To this end, we used cysteine-scanning mutagenesis, positioning single Cys residues at each of 54 different sites of a 222-residue Tim23p construct devoid of native Cys residues (Figure 1A). In vitro–translated [35S]Tim23p monocysteine mutants were integrated into the IM of isolated mitochondria and assembled into functional TIM23 complexes. Endogenous proteins adjacent to specific regions of Tim23p were then identified by chemical cross-linking and immunoprecipitation. Specifically, a homobifunctional thiol-reactive reagent may create a covalent adduct (cross-link) by reacting with the free sulfhydryl group on a given Tim23p monocysteine variant and with a Cys residue on a nearby native protein. Immunoprecipitation of the radiolabeled adduct with antibodies to different TIM23 complex subunits can then be used to identify the cross-linked protein. This approach relies upon the fortuitous proximity and reactivity of Cys groups on the imported [35S]Tim23p mutant and on the endogenous target proteins. However, a positive result, the formation of a covalent linkage, has the advantage of unambiguously identifying the region of proximity between the native subunit and the imported [35S]Tim23p molecule. In this respect, this approach provides the same high-resolution positional information as techniques using site-specific incorporation of photocrosslinking reagents (e.g., (Kanamori et al., 1999 ; McCormick et al., 2003 ; Saksena et al., 2004 ; Davis et al., 2007 ).
A cysteine-less variant of S. cerevisiae Tim23p was prepared by replacement of the native Cys98, Cys209, and Cys213 using site-directed mutagenesis (Figure 1A). As judged by proteolysis experiments with in vitro imported protein (see below), replacement of Cys98 with Ser rendered Tim23p unable to integrate into the IM (data not shown); however, all three positions tolerated replacement with Ala to create a functional Tim23pΔCys, and this construct was used as a background for preparation of all subsequent monocysteine variants.
We first tested whether Tim23pΔCys and Tim23p monocysteine mutants underwent biogenesis similar to that of wild-type Tim23p in isolated mitochondria. Tim23p that is integrated into the IM of intact mitochondria will be protected from added protease. When incubated with intact energized mitochondria, wild-type [35S]Tim23p was integrated into the IM and protected from externally added proteinase K (Figure 1B, lane 2). After osmotic swelling and hypotonic rupture of the OM to produce mitoplasts, the protease degraded the N-terminal region of Tim23p in the IMS and thereby reduced the [35S]Tim23p to the ~14-kDa proteolysis fragment that is diagnostic (Bömer et al., 1997 ; Davis et al., 1998 ) for proper integration (Figure 1B, lane 3, arrow). As controls, incubation of the wild-type construct with de-energized mitochondria prevented integration into the IM (Figure 1B, lane 5), and addition of detergent to solubilize membranes before protease addition rendered the [35S]Tim23p completely sensitive to protease (Figure 1B, lane 4). All mutants were examined with these protease assays, and representative results are shown (Figure 1B) for Tim23pΔCys and mutants with Cys substitutions in TMS1 (F114C), L1 (K131C), and TMS2 (I161C). For all mutants used in this study, the percentage of protein protected from external protease in intact mitochondria was similar (within ~20%) to wild-type, indicating that the Cys substitutions used did not interfere with translocation across the OM. Moreover, the amount of proteolytic fragment after hypotonic swelling was similar in all cases, indicating that none of the Cys substitutions interfered with integration into the IM or with topogenesis.
To determine whether Tim23pΔCys and each monocysteine mutant were properly assembled into TIM23 complexes of isolated mitochondria, we performed coimmunoprecipitation experiments (Bömer et al., 1997 ; Ryan et al., 1998 ). After import into mitochondria and membrane solubilization under nondenaturing conditions that keep the TIM23 complex intact, wild-type [35S]Tim23p was coimmunoprecipitated with antiserum to Tim17p (Figure 1C, lane 2). As negative controls, wild-type [35S]Tim23p incubated with de-energized mitochondria (Figure 1C, lane 4) and the [35S]Tim23p G186D mutant (Lohret et al., 1997 ; Ryan et al., 1998 ; Figure 1C, lane 6) were not precipitated by Tim17p antiserum, thereby demonstrating the requirement of proper integration and assembly for coimmunoprecipitation. Tim23pΔCys and all monocysteine variants exhibited assembly similar to wild type, as demonstrated by representative mutants (Figure 1C, lanes 8, 10, 12, and 14). We conclude that the monocysteine mutants used in this study were able to assemble into TIM23 complexes with efficiency similar to wild type. Notably, the coimmunoprecipitation efficiencies shown here are similar to those obtained previously with in vitro–translated and imported Tim23p (Bömer et al., 1997 ). Although the exact steady-state amount of imported [35S]Tim23p that assembles into a complex cannot be determined by this assay, these results indicate that the monocysteine mutations do not significantly affect Tim23p assembly, and that proximity to other subunits obtained by cross-linking will reflect the native situation.
Having established that the Tim23p monocysteine mutants properly integrated into the IM and assembled into TIM23 complexes, we used these constructs in chemical cross-linking experiments to detect subunits of the endogenous complex that are proximal to different regions of Tim23p. Our primary interest was in identifying cross-linked adducts between the imported Tim23p monocysteine mutants and the essential membrane-bound components of the TIM23 complex (Tim17p, Tim23p, and Tim50p). Figure 2 shows typical results of experiments using the thiol-reactive homobifunctional cross-linking reagent BMOE, which has a spacer arm of 8 Å. As a negative control, imported [35S]Tim23pΔCys did not show any high-molecular-weight adducts after incubation with the chemical cross-linker (Figure 2A, compare lanes 1 and 2), indicating that under the reaction conditions used, there was no spurious cross-linking to non-Cys residues on the imported [35S]Tim32p molecule.
The [35S]Tim23p I111C mutant, with a Cys substitution in TMS1, produced a number of high-molecular-weight adducts after incubation with the cross-linker (Figure 2A, compare lanes 3 and 4). Immunoprecipitation revealed that the adduct with a molecular weight of ~45 kDa was a cross-link between the imported [35S]Tim23p I111C and endogenous Tim17p (Figure 2A, lane 5).
To test for proximity between imported [35S]Tim23p variants and endogenous Tim23p, we used mitochondria isolated from cells expressing Tim23p-HA, a fusion Tim23p protein in which the influenza HA epitope is inserted at the carboxy terminus (Davis et al., 1998 ). Import of [35S]Tim23p monocysteine mutants into these mitochondria, followed by cross-linking and immunoprecipitation with antiserum to the HA tag, was then used to detect proximity to endogenous Tim23p molecules. The [35S]Tim23p N43C mutant, with a Cys substitution in the IMS region, produced a cross-linking adduct of ~60 kDa (Figure 2A, lane 7) that was identified as Tim23p-HA by immunoprecipitation (Figure 2A, lane 8).
The [35S]Tim23p S80C mutant, another construct with an IMS Cys substitution, produced one cross-linking adduct of ~95 kDa and a less intense adduct of ~70 kDa (Figure 2A, lane 10), both of which were identified as cross-links to Tim50p by immunoprecipitation (Figure 2A, lane 11). The apparent molecular mass of the major Tim23p-Tim50p adduct based on this SDS-PAGE analysis is significantly higher than the ~79 kDa expected for a covalent Tim23p-Tim50p adduct. This variation in electrophoretic mobility could be due to the site of covalent linkage between a [35S]Tim23p derivative and endogenous Tim50p, a phenomenon observed in other systems (Plath et al., 1998 ; McCormick et al., 2003 , but see below). Indeed, multiple photoadduct bands have been observed previously between Tim50p and imported Tim23p (Davis et al., 2007 ).
To determine whether imported Tim23p monocysteine mutants were assembled into functional TIM23 complexes, we examined whether the mutants were adjacent to substrate proteins that had been trapped in the TIM23 complex during import. When pSu9-DHFR, a fusion protein between the first 69 amino acids of subunit 9 of the mitochondrial FO-ATPase and mouse dihydrofolate reductase (Rapaport et al., 1998 ), was imported in the presence of its specific ligand MTX, the resultant tightly folded DHFR-MTX domain could not cross the TOM complex channel and therefore formed a translocation intermediate in which the substrate presequence was engaged with the TIM23 complex (Eilers and Schatz, 1986 ). Thus, [35S]Tim23p S34C, a mutant with a Cys substitution in the N-terminal IMS region, was first imported into mitochondria. Purified pSu9-DHFR was then incubated with those mitochondria in the presence of MTX, after which cross-linker was added (Figure 2B). Although several proteins were cross-linked to imported [35S]Tim23p S34C, an adduct of ~45 kDa was observed with pSu9-DHFR M15C (bearing a single Cys residue at position 15 of the presquence; Figure 2B, lane 4), but not with pSu9-DHFR ΔCys (a version of the fusion protein lacking Cys residues in the presequence; Figure 2B, lane 3). This band was identified as a cross-link between pSu9-DHFR and newly imported [35S]Tim23p because the radioactive species was immunoprecipitated with pentahistidine antiserum that recognizes the hexahistidine affinity tag on the pSu9-DHFR protein (Figure 2B, lanes 5 and 6). Identical results were obtained when the sample was immunoprecipiated with DHFR antiserum (data not shown). Taken together, these data show that the trapped substrate cross-links to the Tim23p mutant via its Cys residue, and hence confirm that the imported Tim23p monocysteine mutants are assembled into functional, translocation-competent TIM23 complexes.
We expanded our cross-linking analysis to explore regions of proximity between our library of Tim23p monocysteine mutants and endogenous Tim17p, Tim23p-HA, and Tim50p. We found robust cross-linking adducts between endogenous Tim17p and imported [35S]Tim23p mutants that had Cys substitutions in TMS1 (Figure 3A). Because S. cerevisiae Tim17p contains two Cys residues in IMS-facing loops and two Cys residues predicted to be within the membrane (Tim17p TMS4, Figure 1A), Cys residues in Tim23p TMS1 and Tim17p TMS4 are likely to be proximal, provided that the local environment is conducive to the cross-linking reaction (see Discussion). These cross-linking results are also consistent with the previously observed coimmunoprecipitation of Tim17p with the C-terminal (membrane-embedded) domain of Tim23p (Ryan et al., 1998 ). A helical wheel projection showing Tim23p TMS1 sites that cross-link to Tim17p (Figure 3D) indicates that cross-linking to Tim17p occurs on opposite sides of the Tim23p TMS1 helix. These data suggest that multiple copies of Tim17p associate with Tim23p on opposite faces of TMS1 and/or that TMS1 of Tim23p has significant rotational flexibility, but exists stably in only a few orientations. Whatever the case, these results demonstrate both the high resolution of this cross-linking approach and also the specificity of the quaternary arrangement of proteins in the TIM23 complex.
Cross-links to endogenous Tim23p-HA were primarily detected with imported [35S]Tim23p mutants having Cys substitutions in the IMS region. In addition, significant adduct formation occurred at the IMS-facing L2 and at position 220 near the C-terminus, along with a few weak adducts detected at other sites throughout the protein (Figure 3B). Because the native Cys residues in endogenous Tim23p of S. cerevisiae include a single Cys located in the IMS just N-terminal to TMS1 and two Cys in TMS4 (Figure 1A), the most likely cross-linking partner for the N-terminal [35S]Tim23p mutants is the IMS-facing Cys residue in Tim23p-HA, especially given the short span of the BMOE cross-linker and the extensive range over which cross-linking occurred. Proximity between the IMS regions of Tim23p assembled into a TIM23 complex is consistent with previous work showing that dimerization of Tim23p molecules occurs via the second half of the N-terminal domains (Bauer et al., 1996 ). But the similarity in cross-linking yields for all sites tested in the mutant Tim23p IMS domain strongly indicates that the N-terminal half of Tim23p does not occupy a fixed position in the TIM23 complex. Instead, the IMS domain appears flexible and free to move such that residues from 4 to 94 on the imported mutants are able to contact Cys98 of the endogenous Tim23p during the course of the cross-linking reaction.
Only Tim23p monocysteine mutants with Cys substitutions in the IMS and in TMS1 were used to test for proximity to native Tim50p. Of the IMS sites, only Cys residues located in the C-terminal part of this domain (positions 80 and 94) yielded robust cross-linked adducts (Figure 3C). This result is consistent with previous observations that the IMS domain of Tim50p directly interacts with the C-terminal half of the IMS domain of Tim23p (Geissler et al., 2002 ; Yamamoto et al., 2002 ; Mokranjac et al., 2003 ). In addition, strong cross-linking was detected between TMS1 of Tim23p and Tim50p (Figure 3C), and a helical projection indicates that this cross-linking occurs over a broad arc of the TMS1 helix (Figure 3D). Given that S. cerevisiae Tim50p has only a single Cys (Figure 1A), the cross-linking partners can be unambiguously identified: Cys sites in the extreme C-terminal IMS region and in TMS1 of Tim23p are proximal to Cys286 of Tim50p. Because each protein bears a single Cys, a single band representing the Tim23p-Tim50p adduct would be expected to be immunoprecipitated with antiserum to Tim50p. The presence of multiple bands seen here in certain cases may, for example, suggest partial proteolysis of the sample during immunoprecipitation. Notably, the extensive and overlapping cross-linking of Tim17p and Tim50p with Tim23p TMS1 suggests that an imported Tim23p exists in multiple different states within the TIM23 complex and/or that Tim23p has sufficient conformational flexibility to position TMS1 proximal to either Tim17p or Tim50p (see Discussion).
It is important to note that using homobifunctional reagents with the same maleimide reactive groups, but with longer spacer arm lengths (up to 16.1 Å), did not yield any qualitative differences in the cross-linking profiles reported here.
Isolated Tim23p and the TIM23 complex display voltage gating behavior (Lohret et al., 1997 ; Truscott et al., 2001 ). Other channels, such as voltage-gated potassium channels, undergo conformational changes and transmembrane movements to varying extents in response to changing electric field across the membrane (Tombola et al., 2005 ). We therefore examined whether our cross-linking strategy would detect changes in TIM23 complex quaternary structure if the Δψ across the IM changed. In this approach, the energized import of a given [35S]Tim23p monocysteine mutant was followed by the addition of different uncoupling agents and ionophores to dissipate the Δψ before cross-linking.
Collapse of the Δψ by the K+ ionophore valinomycin or by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) specifically blocked cross-linking between [35S]Tim23p I111C and Tim17p (Figure 4A, compare lane 1 with lanes 2 and 3 [totals] and lane 4 with lanes 6 through 10 [immunoprecipitations]). Indeed, dissipation of the Δψ by CCCP completely blocked cross-linking between all TMS1 Cys sites and native Tim17p (data not shown). In contrast, nigericin, which catalyzes the electroneutral exchange of H+ and K+ and thus collapses only the pH gradient (believed to constitute a minor fraction of the protonmotive force across the IM; Nicholls and Ferguson, 1992 ), caused only a small change in [35S]Tim23p I111C-Tim17p cross-linking (Figure 4A, lane 5). These results therefore show that the proximity of residues in Tim23p TMS1 and Tim17p is strongly dependent on the Δψ across the IM. It should be noted, however, that the Tim23p-Tim17p core complex has been shown to remain stable after dissipation of the Δψ by blue native PAGE (Dekker et al., 1997 ). Therefore, any interactive surface(s) between these two subunits that remain(s) after Δψ dissipation likely exists between Tim23p and a site on Tim17p that lacks a nearby Cys (and therefore any potential cross-linking site in our experiments).
Cross-linking between [35S]Tim23p mutants and endogenous Tim23p-HA varied with the electrochemical gradient in a different way (Figure 4B). [35S]Tim23p K66C, bearing a Cys substitution in the C-terminal half of the IMS region, shows only a slight valinomycin-dependent decrease in cross-linking to Tim23p-HA (Figure 4B, compare lanes 4 and 6) and only a slight decrease upon titration with CCCP (Figure 4B, compare lanes 4 and lanes 8–10). However, cross-linking between [35S]Tim23p K66C and endogenous Tim23p-HA was significantly reduced with the inclusion of antimycin A (an inhibitor of complex III of the respiratory chain) and oligomycin (an inhibitor of the FO/F1-ATPase; Figure 4B, lane 7). A separate series of experiments revealed that the presence of 4 μM antimycin A alone reduced the extent of cross-linking between [35S]Tim23p K66C and Tim23p-HA to 43% of the control value. Qualitatively similar patterns were observed when [35S]Tim23p S30C and T94C cross-linking to Tim23p-HA was analyzed (data not shown). Interestingly, the presence of potassium cyanide (an inhibitor of complex IV) did not reduce the efficiency of cross-linking between Tim23p IMS regions (data not shown), indicating a specific effect of antimycin A that is not simply attributable to reduced Δψ nor to a general block of respiratory electron flow. We note also that these drugs can affect matrix ATP levels and thus our observed effects may be due in part to altered ATP concentrations.
In dramatic contrast to the above results, the extent of adduct formation between [35S]Tim23p S80C and native Tim50p unexpectedly increased in the presence of the valinomycin and nigericin ionophores (Figure 4C, lanes 5 and 6) and with increasing concentrations of the uncoupler CCCP (Figure 4C, lanes 8–10), consistent with a greater degree of proximity between the two subunits as Δψ decreases. The presence of 4 μM antimycin A alone increased cross-linking efficiency between [35S]Tim23p S80C and Tim50p by 35% (data not shown). Thus, the structural arrangement of Tim23p within the TIM23 complex is significantly altered with respect to Tim17p, Tim50p, and other Tim23p molecules when the Δψ is dissipated.
To test whether the observed Δψ-dependence of cross-linking efficiency was reversible, we counteracted the CCCP-induced Δψ collapse by washing the mitochondria with DTT and respiratory substrate (Yaffe, 1991 ). This same strategy was used previously to show that Δψ-dependent import of polytopic IM proteins can be restored after CCCP incubation and that such precursors are in fact productive on-pathway intermediates (Ryan et al., 1999 ; Davis et al., 2000 , 2007 ). Thus, mitochondria were incubated with CCCP and then given a second wash, either with CCCP to keep the Δψ dissipated or with NADH and DTT to reestablish the Δψ before the cross-linking reaction (Figure 5). As shown above, incubation with CCCP reduced the cross-linking of [35S]Tim23p mutants I111C and K66C to Tim17p and to Tim23p-HA, respectively, and enhanced cross-linking between [35S]Tim23p S80C and Tim50p all to extents similar to those seen in Figure 4 (Figure 5, compare A and B samples). But when CCCP-treated mitochondria were incubated with NADH and DTT before cross-linking, the extents of the cross-linking were very similar to those of non-CCCP–treated samples for all mutants (Figure 5, compare A and C samples). The Δψ-dependent changes in cross-linking observed in this study are therefore reversible and likely reflect bona fide changes in subunit proximity that occur with changes in the energetic state of the IM.
Because changes in Δψ effected such profound changes in subunit proximity, particularly for the Tim23p IMS and TMS1 sites, we examined whether changes in the Δψ could also alter the accessibility of these sites to a large hydrophilic reagent. Both energized and valinomycin-treated mitochondria containing an imported [35S]Tim23p monocysteine mutant were incubated with the large membrane-impermeant sulfhydryl reactive reagent IASD (Figure 6). IASD labeling can be detected by a shift in the electrophoretic mobility of the protein in SDS-PAGE (Krishnasastry et al., 1994 ). Under the reaction conditions used, the [35S]Tim23p S80C mutant, with a Cys residue in the IMS, was accessible to IASD in both fully energized and valinomycin-treated mitochondria, whereas the [35S]Tim23p L157C mutant, with a Cys in TMS2, was not labeled by IASD under either condition (Figure 6, compare lanes 2 and 3 with 1). In contrast, [35S]Tim23p mutants I111C and F114C, with Cys residues in TMS1, displayed Δψ-dependent IASD labeling. These TMS1 sites were partially accessible to IASD in fully energized mitochondria (Figure 6, lane 2) but the extent of IASD labeling increased significantly after valinomycin treatment (Figure 6, lane 3). Therefore, collapse of the Δψ increased the exposure of these TMS1 sites to an aqueous compartment contiguous with the IMS.
Finally, we tested whether the presence of substrate in the TIM23 complex might alter the cross-linking of its subunits. After the import of [35S]Tim23p monocysteine mutants, mitochondria were washed and supplied with pSu9-DHFR at different concentrations (up to 2 μM) in the presence of MTX to trap the substrate in the TIM23 complex. By using a Cys-less pSu9-DHFR substrate, no cross-linking between the overexpressed substrate and assembled [35S]Tim23p molecules could occur (Figure 2B). Given the estimated Tim23p concentration of 20 pmol per mg of mitochondrial protein (Dekker et al., 1997 ; Moro et al., 1999 ), substrate was added up to a roughly 200-fold molar excess over Tim23p molecules.
The extent of cross-linking between [35S]Tim23p I111C and endogenous Tim17p decreased dramatically when increasing amounts of pSu9-DHFR engaged the TIM23 complex (Figure 7A). Interestingly, cross-linking between Cys residues in the [35S]Tim23p IMS region and Tim23p-HA varied with pSu9-DHFR-MTX concentration in a position-dependent manner. Cross-linking between Tim23p-HA and [35S]Tim23p S30C, with a Cys in the N-terminal half of the IMS, was not strongly dependent on the presence of substrate (Figure 7B, ■). In contrast, cross-linking between Tim23p-HA and [35S]Tim23p S80C, with a Cys in the C-terminal half of the IMS, decreased significantly with increasing substrate concentration (Figure 7B, □). Thus, the cross-linking efficiencies between Tim23p TMS1 and Tim17p and between the C-terminal half of the Tim23p IMS region and other Tim23p subunits were significantly reduced by the presence of a translocation intermediate.
This comprehensive and high resolution study provides five main insights into the quaternary structure of the TIM23 complex. First, after importing each of 54 different Tim23p monocysteine mutants into intact and energized mitochondria, specific regions of a Tim23p molecule were shown to be adjacent to Tim17p, Tim50p, and at least one other Tim23p protein in a fully assembled and functional TIM23 complex (see below). Second, the structural proximity and hence arrangement of proteins within the TIM23 complex is dependent on the Δψ. Third, the Δψ-dependent conformational rearrangements are reversible, suggesting that they represent a true physiological response to the energized state of the IM. Fourth, different protein–protein proximities in the TIM23 complex are detected when a substrate is trapped inside the translocase, and these changes reflect Δψ-dependent changes. Fifth, these combined results show that an assembled TIM23 complex exists in a minimum of three states (energized, de-energized, and energized with substrate) within the IM that differ both structurally and functionally. The TIM23 complex is therefore a dynamic multicomponent assembly whose functional state is dictated, and presumably regulated, by changes in quaternary structure in response to cellular conditions.
Tim23p TMS1 is adjacent to Tim17p (Figures 3A and and8A)8A) because nine residues of TMS1 (from Tyr105 to Phe114) cross-linked to Tim17p. Interestingly, these sites are found on two oppositely oriented faces of the TMS1 helix when mapped onto a helical projection (Figure 3D). Although rotational degeneracy of a transmembrane helix in the TIM23 complex could explain such differential cross-linking, the specificity with which cross-linked adducts were formed on the two faces strongly suggests that TMS1 of Tim23p contacts two separate Tim17p molecules on opposite sides. Given the topological placement of native Cys residues within Tim17p (Figure 8A), its two Cys residues in the N-terminal part of TMS4 are the most likely cross-linking partners for Tim23p TMS1 monocysteine mutants.
Among the TIM23 complex structural relationships characterized, the juxtaposition of Tim23p TMS1 and Tim17p was the most sensitive both to changes in Δψ (Figure 4A) and to the presence of substrate (Figure 7). It is not clear whether the Δψ- and substrate-dependent changes in Tim23p TMS1-Tim17p cross-linking have the same molecular basis (i.e., whether the collapse of the Δψ or the addition of substrate elicit the same conformational changes in the TIM23 complex). But whatever the origin, this dynamic Δψ- and substrate-dependent proximity may very well be related to the recently identified role of Tim17p in regulating the substrate-dependent voltage gating of Tim23p (Meier et al., 2005 ; Martinez-Caballero et al., 2007 ).
The Tim23p IMS region is proximal to other Tim23p subunits in the TIM23 complex (Figures 3B and and8B).8B). The wide range of sites in the [35S]Tim23p mutants that cross-link to native Tim23p presumably through its single Cys in the IMS indicates a high degree of Tim23p conformational flexibility in the IMS. A decrease in the Δψ leads to a modest reduction in cross-linking between Tim23p IMS sites and other Tim23p subunits (Figure 4B), whereas the presence of a translocation intermediate significantly reduces cross-linking between Tim23p sites in the C-terminal half of the IMS region and other Tim23p subunits (Figure 7B). These results are consistent with previous work, indicating that Tim23p dimerizes via a putative leucine zipper structure in the C-terminal half of its IMS domain in a manner dependent on the functional state of the channel: dimer formation requires the Δψ, and matrix targeting presequences stimulate dimer dissociation (Bauer et al., 1996 ). Finally, the strong effect of antimycin A on Tim23p IMS cross-linking (Figure 4B) could result in part from the recently described coupling between Tim21p of the TIM23 complex and respiratory complexes III-IV (van der Laan et al., 2006 ). Alternatively, the effect could arise from the binding of antimycin A to a site other than complex III (e.g., Tzung et al., 2001 ).
The extreme C-terminal end of the IMS region of Tim23p is proximal to Tim50p (Figures 3C and and8C),8C), consistent with previous work showing an interaction between the IMS region of Tim50p and the C-terminal half of the Tim23p IMS domain (Geissler et al., 2002 ; Yamamoto et al., 2002 ; Mokranjac et al., 2003 ). In addition, we unexpectedly found cross-links between Tim50p and TMS1 of Tim23p (Figures 3C and and8C).8C). Given that the putative cross-linking site on Tim50p (Cys268 in the mature protein) is in the C-terminal IMS domain, this suggests that either this region of Tim50p protrudes into the bilayer and/or that Tim23p has the conformational freedom to move TMS1 into the aqueous IMS to some degree. In any case, a helical projection of the sites that cross-link Tim23p TMS1 to Tim50p (Figure 3D) suggests that, in contrast to well-defined sites that cross-link Tim23p TMS1 to Tim17p (Figure 3D), there is significant rotational freedom between TMS1 and Tim50p such that cross-linking occurs over most of the helical circumference.
The cross-linking observed between Tim23p and Tim50p may also provide some insight into the functional role of Tim50p. The reactive Cys in Tim50p falls within a region of the IMS domain that is homologous to NIF/CDCc domains of CTD phosphatases (residues 165-344; Geissler et al., 2002 ; Mokranjac et al., 2003 ). Although the possible role of this domain in Tim50p function is not known, the potential specific interaction between Tim23p TMS1 and this region may provide clues to its function. Our unexpected observation that cross-linking between the IMS region of Tim23p and Tim50p increased when the Δψ collapsed suggests that Tim23p-Tim50p proximity and/or affinity increases upon loss of the Δψ. When coupled with the recent observation that the IMS region of Tim50p maintains the permeability barrier across the IM by promoting the closed state of the Tim23p channel (Meinecke et al., 2006 ), a regulatory mechanism is suggested. Specifically, the closer interaction between Tim23p and Tim50p that we detected upon lowering the Δψ may have evolved to maintain the IM permeability barrier and prevent unregulated collapse of IM gradients.
Our comprehensive cross-linking study has therefore provided unprecedented resolution in characterizing the proximity between TIM23 complex subunits, thereby significantly extending our understanding of the TIM23 complex quaternary structure. Although studies describing protein interactions based on coimmunoprecipitation cannot distinguish between direct contact and association through an intermediary subunit, a covalent bond that directly cross-links two macromolecules shows unambiguously that residues from each protein were adjacent. Moreover, the formation of covalent adducts usually identifies protein surfaces that interact for functional or structural reasons. But even though a lack of cross-linking cannot be taken as evidence for lack of proximity, the cross-linking approach used here maps the proximity of specific helical faces to other proteins at high resolution.
Several models could account for the multiple Δψ- and substrate-induced conformational changes in quaternary structure characterized here. Two possibilities are depicted in Figure 8D. Partial de-insertion of TMS1 from the membrane (case 1) would require that the energetic penalties of moving nonpolar residues into the aqueous phase and disrupting any associative energies with other TMSs in the membrane be offset by some compensatory binding energy (e.g., binding to Tim50p). Alternatively, loss of membrane potential and/or substrate addition may, for example, induce helix tilting and expansion of an aqueous crevice in this region (case 2). Future work will determine to what extent either model, or some combination thereof, is correct. But the conformational changes characterized by this study have provided insight into the Δψ- and substrate-dependent dynamics of the TIM23 complex and hence will direct further investigations.
We are grateful to Yiwei Miao and Yuanlong Shao for outstanding technical assistance and to the members of the Johnson lab for helpful discussions. This work was supported by National Institutes of Health Grant GM26494 (A.E.J.), National Research Service Award GM70266 (N.N.A.), and by the Robert A. Welch Foundation (Chair Grant BE-0017).
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-07-0669) on October 24, 2007.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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