plasmids , strains , and chemicals  all dna manipulations were 
  performed according to standard methods 
  ( 52 ) and all media were 
  prepared by standard procedures 
  ( 53 ) . ( swampscott , ma ) , or fisher scientific ( pittsburgh , pa ) unless otherwise 
  noted . w303 ( acy233 ) 
  mat ura3 leu2 trp1 his3nab2 ( acy427 ) 
  mata leu2 ura3 his3 nab2::his3 ( pac636 ) 
  72nab2mex67 ( swy3601 ) 
   mata trp1 leu2 his3 ura3 nab2::his3 
  mex67::kan ( prs314-mex67 - 5 ) 
  70nab2yra1 ( fsy2327 ) 
  mata ura3 his3 leu2 trp1 yra1::his3 
  nab2::his3 ( pfs1876 , pac636 ) 
   f. stutz 
   pgex4t-3 
  gst , ampr , bacterial expression vector 
   ge healthcare 
   pac1340 
  gst - ct - mlp1 ( residues 14901875 ) in pgex4t-3 , 
  ampr38 pac1678 
  gst - ct5 ( residues 14901585 ) in pgex4t-3 , 
  ampr this study 
   pac1679 
  gst - ct4 ( residues 14901682 ) in pgex4t-3 , 
  ampr this study 
   pac1680 
  gst - ct2 ( residues 14901779 ) in pgex4t-3 , 
  ampr this study 
   pac1681 
  gst - ct6 ( residues 15861779 ) in pgex4t-3 , 
  ampr this study 
   pac1723 
  gst - ct7 ( residues 16771779 ) in pgex4t-3 , 
  ampr this study 
   pgex4t-1 
  gst , ampr , bacterial expression vector 
   ge healthcare 
   pac2058 
  gst - nab2-n - wt ( residues 197 ) in pgex4t-1 , 
  ampr49 pac2349 
  gst - nab2-n - f72d ( residues 197 ) in pgex4t-1 , 
  ampr49 pac2350 
  gst - nab2-n - f73d ( residues 197 ) in pgex4t-1 , 
  ampr49 pac2453 
  gst - nab2-n - f73w ( residues 197 ) in pgex4t-1 , 
  ampr49 pet28a 
  his6 , kanr , bacterial expression vector 
   novagen 
   pac785 
  his6-nab2 in pet28a , kanr32 pet30a 
  his6 , s - tag , kanr , bacterial expression vector 
   novagen 
   pac2052 
  his6-nab2-n - wt ( residues 197 ) in pet30a , 
  kanr this study 
   pac2054 
  his6-s - tag - mlp1-nbd ( residues 15861768 ) in 
  pet30a , kanr this study 
   pps295 ( pac18 ) 
  pgal1 , 2 , ura373 pac2075 
  pgal1-sv40 bipartite nls - mlp1-nbd ( residues 
  15861768 ) , 2 , ura3 , ampr this study 
   pac1690 
  pgal1-sv40 bipartite nls - myc - ct - mlp1 ( residues 
  14901875 ) , 2 , ura3 , ampr this study 
   pac1692 
  pgal1-sv40 bipartite nls - myc - ct5 ( residues 14901585 ) , 
  2 , ura3 , ampr this study 
   pac1693 
  pgal1-sv40 bipartite nls - myc - ct4 ( residues 14901682 ) , 
  2 , ura3 , ampr this study 
   pac1694 
  pgal1-sv40 bipartite nls - myc - ct3 ( residues 14901717 ) , 2 , 
  ura3 , ampr this study 
   pac1695 
  pgal1-sv40 bipartite nls - myc - ct2 ( residues 14901779 ) , 2 , 
  ura3 , ampr this study 
   pac1696 
  pgal1-sv40 bipartite nls - myc - ct1 ( residues 14901811 ) , 
  2 , ura3 , ampr this study 
   pac636 
  nab2 , cen , ura3 , ampr32 pac717 
  nab2 , cen , leu2 , ampr36 pac1152 
  nab2 n , cen , leu2 , ampr36 pac2351 
  nab2 f72d , cen , leu2 , ampr this study 
   pac2352 
  nab2 f73d , cen , leu2 , ampr this study 
   pac1036 
  rgg nab2-gfp , cen , leu2 , ampr36 pfs1876 
  yra1 , cen , ura3 , ampr19 pfs2152 ( pac2266 ) 
  yra18 , cen , trp1 , ampr21 
  prs314-mex67 - 5mex67 - 5 , cen , trp1 , ampr8 yeast strains and plasmids generation of nab2 mutants  nab2 mutants gst - nt f72d 
  ( pac2349 ) , gst - nt f73d ( pac2350 ) , and gst - nt - f73w ( pac2453 ) were previously 
  described ( 49 ) . nab2 mutants 
  nab2 f72d ( pac2351 ) and nab2 f73d ( pac2352 ) were generated 
  by site - directed mutagenesis using oligonucleotides ( integrated dna 
  technologies ) encoding the f72d or f73d amino acid substitution , nab2 
  ( pac717 ) plasmid template , and quikchange site - directed mutagenesis kit 
  ( stratagene ) . all constructs were sequenced to ensure the presence of each 
  desired mutation and the absence of any additional mutations . protein expression and purification  recombinant gst fusion 
  and his - tagged proteins were expressed in bacteria and purified for in 
  vitro solution and solid phase binding assays . gst ( pgex4t-3 ) , 
  gst - ct - mlp1 ( pac1340 ) , gst - ct2 ( pac1680 ) , gst - ct4 ( pac1679 ) , gst - ct5 
  ( pac1678 ) , gst - ct6 ( pac1681 ) , gst - ct7 ( pac1723 ) , and his - nab2 ( pac785 ) were 
  expressed in escherichia coli de3 cells and purified by batch 
  purification . gst ( pgex4t-1 ) , gst - nab2-n - wt ( pac2058 ) , gst - nab2-n - f72d 
  ( pac2349 ) , gst - nab2-n - f73d ( pac2350 ) , gst - nab2-n - f73w ( pac2453 ) , his - nab2-n - wt 
  ( pac2052 ) , and his - s - tag - mlp1-nbd ( pac2054 ) were expressed in e. coli 
  de3 cells and purified by column chromatography . overnight cultures were used 
  to inoculate 100 ml of lb media for batch purification or 1 liter of lb media 
  for purification by column chromatography . cultures were grown at 37 c to 
  an a600 of 0.60.8 , induced with 200 
  m isopropyl 1-thio - d - galactopyranoside , and grown at 
  30 c for 5 h. for batch purification of gst fusion proteins , cells were 
  collected and lysed in 10 ml of phosphate - buffered saline ( pbs ) supplemented 
  with protease inhibitor mixture ( 1 mm phenylmethylsulfonyl 
  fluoride , 3 ng / ml pepstatin a , leupeptin , aproptinin , and chymostatin ) by 
  incubation with lysozyme ( 100 mg / ml ) for 30 min on ice and sonication . lysates 
  were cleared by centrifugation and incubated with glutathione - sepharose 4b ( ge 
  healthcare ) for 2 h at 4c with mixing . the beads were then washed once 
  with 10 ml of pbs supplemented with 0.5% triton x-100 and twice with 10 ml of 
  pbs . for purification of gst fusion proteins by column chromatography , cells 
  were collected and lysed in 20 ml of buffer a ( pbs , 2 mm 
  2-mercaptoethanol ) supplemented with protease inhibitor mixture by a french 
  press and cleared lysates were loaded onto a gstrap 4b column ( ge healthcare ) 
  pre - equilibrated with buffer a. bound protein was eluted with a linear 
  gradient of buffer b ( pbs , 2 mm 2-mercaptoethanol , 10 mm 
  reduced glutathione ) and fractions containing gst protein were dialyzed into 
  pbs supplemented with 2 mm dithiothreitol ( dtt ) . for batch 
  purification of histagged proteins , cells were collected and lysed in 10 ml of 
  lysis buffer ( 50 mm nah2po4 , ph 7.4 , 300 
  mm nacl , 10 mm imidazole ) supplemented with protease 
  inhibitor mixture by incubation with lysozyme and sonication . lysates were 
  cleared by centrifugation and incubated with nickel - nitrilotriacetic 
  acid - agarose ( qiagen ) in lysis buffer for 2 h at 4 c with mixing . the 
  beads were then washed twice with 10 ml of wash buffer ( 50 mm 
  nah2po4 , ph 7.4 , 300 mm nacl , 20 
  mm imidazole ) . his - tagged proteins were eluted from agarose with 1 
  ml of elution buffer ( 50 mm nah2po4 , ph 7.4 , 
  300 mm nacl , 250 mm imidazole ) and buffer exchanged into 
  pbs supplemented with 2 mm dtt using a centricon centrifugal filter 
  device ( millipore ) . for purification of his - tagged proteins by column 
  chromatography , cells were collected and lysed in 20 ml of buffer a ( 50 
  mm nah2po4 , ph 7.4 , 500 mm nacl , 
  20 mm imidazole , 2 mm 2-mercaptoethanol ) supplemented 
  with protease inhibitor mixture by a french press and cleared lysates were 
  loaded onto a hitrap imac hp column ( ge healthcare ) pre - equilibrated with 
  buffer a. bound protein was eluted with a linear gradient of buffer b ( 50 
  mm nah2po4 , ph 7.4 , 300 mm nacl , 
  250 mm imidazole , 2 mm 2-mercaptoethanol ) and fractions 
  containing his - tagged protein were dialyzed into pbs supplemented with 2 
  mm dtt . solution binding assays  for nab2 binding to ct - mlp1 and 
  ct - mlp1 truncation mutants , sepharose - bound gst , gst - ct - mlp1 , or gst - ct2 and 
  -47 ( 6 g ) was incubated with 2 g of purified his - nab2 at 4 
  c in 1 ml of pbs supplemented with protease inhibitor mixture , 0.1 mg / ml 
  bsa competitor , and 0.5% triton x-100 for 1 h 30 min at 4c with mixing . unbound fractions were collected and the beads were washed three times with 1 
  ml of pbs for 5 min each . bound fractions were eluted with loading buffer and 
  samples were analyzed by sds - page followed immunoblotting with an anti - his 
  antibody ( santa cruz biotechnology , santa cruz , ca ) . the intensities of the 
  his - nab2 bands in the bound fractions were quantitated by integration of the 
  pixel densities of the bands using imagej version 1.4 software ( national 
  institute of health , md ; 
  rsb.info.nih.gov/ij/ ) 
  and percentage bound relative to ct - mlp1 was calculated by dividing the 
  his - nab2 band intensity in each bound fraction by the his - nab2 band intensity 
  in the bound fraction of gst - ct - mlp1 . for mlp1-nbd binding to nab2-n , 
  his - nab2-n - wt , or ovalbumin coupled to cnbr - sepharose beads ( 30 l ) , as 
  described previously ( 49 ) , was 
  incubated with purified his - mlp1-nbd ( 20 g ) in 1 ml of pbs supplemented 
  with protease inhibitor mixture , 2 mm dtt , and 0.5 mg / ml bsa for 1 
  h 30 min at 4 c with mixing . unbound fractions were collected and the 
  beads were washed three times with 1 ml of pbs for 5 min each . bound fractions 
  were eluted with loading buffer and samples were analyzed by sds - page followed 
  by coomassie staining . for co - purification of mlp1-nbd with nab2-n , 50-ml 
  cultures of de3 cells expressing gst - nab2-n or his - mlp1-nbd were combined , 
  collected , and lysed in 10 ml of pbs supplemented with protease inhibitor 
  mixture by sonication . lysates were cleared and incubated with 
  glutathione - sepharose for 2 has described previously for batch purification of 
  gst fusion proteins . the beads were washed three times with 5 ml of pbs for 2 
  min each . unbound , bound , and wash samples were analyzed by sds - page followed 
  by coomassie staining . solid phase binding assays were 
  performed essentially as described by bayliss et al . microtiter immulux hb 
  plates ( dynex ) were coated with 100 l / well of 125 nm gst , 
  gst - nab2-n ( residues 197 ) , gst - nab2-n - f72d , gst - nab2-n - f73d , or 
  gst - nab2-n - f73w in coating buffer ( pbs supplemented with 2 mm dtt 
  and 0.2 mm phenylmethylsulfonyl fluoride ) for 16 h at 4 c on a 
  rocker . after adsorption , plates were washed three times by immersion in pbs 
  and incubated in 100 l of binding buffer ( coating buffer supplemented with 
  3% bsa and 0.1% tween 20 ) overnight at 4 c on a rocker . binding reactions 
  were carried out for 2 h at 4 c with 100 l / well of 15 
  m s - tag - mlp1-nbd ( residues 15861768 ) protein in binding 
  buffer . after binding , plates were washed three times by immersion in binding 
  buffer without bsa and proteins were cross - linked for 15 min at room 
  temperature by incubation in 1 mg / ml 
  1-ethyl-3-(3-dimethyl - aminopropyl)carboiimide ( pierce ) in the same buffer . the 
  wells were then washed for 20 min in pbs - t ( pbs supplemented with 0.2% tween 
  20 ) , 10 min with pbs - t containing 100 mm ethanolamine , and finally 
  incubated for 10 min in pbs - t containing 3% bsa . the bound s - tagged proteins 
  were detected by incubation with s - protein - horseradish peroxidase conjugate 
  ( novagen ) in coating buffer containing 1% bsa and 0.1% tween 20 for 1 h at 
  4c on rocker . the plates were washed three times by immersion in pbs . 
  then , 100 l / well of horseradish peroxidase substrate ( 1-step turbo 
  tmb ( 3,35,5-tetramethylbenzidine)-elisa ( pierce ) ) was added for 
  30 min at room temperature and the reaction was stopped by addition of 100 
  l of 2 m h2so4 . the absorbance of the 
  samples was measured at 450 nm with an elx808 ultra microplate 
  reader with kcjunior software ( bio - tek instruments , inc . ) . for each plate , average absorbance values at a450 were determined for gst 
  and gst - nab2-n proteins at each s - tag - mlp1-nbd concentration from triplicate 
  samples . background gst absorbance values were subtracted from those of the 
  gst - nab2 proteins . from three plates , absorbance values for gst - nab2-n 
  proteins at each s - tag - mlp1-nbd concentration were used to generate saturation 
  binding curves by non - linear regression using prism 4 software ( graphpad 
  software , inc . ) and calculate the mean apparent kd . standard error of the mean ( s.e . ; standard deviation divided by the square 
  root of n , where n = 3 ) values of a450 
  readings from triplicate samples of gst - nab2-n proteins at each s - tag - mlp1-nbd 
  concentration for binding curves in fig . values of triplicate mean 
  kd values for gst - nab2-n protein binding to s - tag - mlp1-nbd 
  in table 2 were calculated 
  using prism 4 software . table 2dissociation constants for nab2-n wild - type / mutants and mlp1-nbd 
  data represent the kd means  s.e . from triplicate experiments.nab2-nmlp1kd apparent m gst - nab2-n - wt 
   mlp1-nbd 
   1.26  0.38 
   gst - nab2-n - f72d 
   mlp1-nbd 
   2.31  1.06 
   gst - nab2-n - f73d 
   mlp1-nbd 
   not detectable 
   gst - nab2-n - f73w 
   mlp1-nbd 
   0.83  0.18 
 
  gst 
 
  mlp1-nbd 
 
  not detectable 
   dissociation constants for nab2-n wild - type / mutants and mlp1-nbd 
  data represent the kd means  s.e . from triplicate 
  experiments . rgg - nab2-gfp relocalization assay  to examine the 
  effect of mlp1 expression on nab2 localization in yeast cells , we exploited a 
  previously developed assay where relocalization of a largely cytoplasmic nab2 
  mutant fused to gfp , rgg - nab2-gfp , to the nucleus is monitored upon 
  mlp1 expression ( 38 ) . for 
  analysis of the effect of expression of ct - mlp1 , ct - mlp1 truncation mutants 
  ct15 or mlp1-nbd on the localization of rgg - nab2-gfp , wild - type 
  w303 cells expressing rgg - nab2-gfp ( pac1036 ) and containing 
  galactose - inducible ct - mlp1 ( pac1690 ) , ct1 ( pac1696 ) , 
  ct2 ( pac1695 ) , ct3 ( pac1694 ) , ct4 ( pac1693 ) , 
  ct5 ( pac1692 ) , mlp1-nbd ( pac2075 ) , or vector control 
  ( pps295 ) were grown in minimal media with 2% glucose overnight at 30 c , 
  transferred to minimal media with 2% galactose , and grown to log phase at 30 
  c . for all samples , the localization of rgg - nab2-gfp was examined 
  by direct fluorescence microscopy . fluorescence in situ hybridization ( fish)the intracellular 
  localization of poly(a ) rna was assayed by fish 
  ( 32 ) . briefly , wild - type w303 
  cells containing galactose - inducible ct - mlp1 ( pac1690 ) , ct1 
  ( pac1696 ) , ct2 ( pac1695 ) , ct3 ( pac1694 ) , ct4 
  ( pac1693 ) , ct5 ( pac1692 ) , mlp1-nbd ( pac2075 ) , or 
  vector control ( pps295 ) were grown in minimal media with 2% glucose overnight 
  at 30 c , transferred to minimal media with 2% galactose , and grown to log 
  phase at 30 c . mex67 - 5 cells containing nab2 ( pac717 ) , nab2 
  n ( pac1152 ) , nab2 f72d ( pac2351 ) , or nab2 f73d ( pac2352 ) were grown in minimal media with 2% glucose overnight , 
  transferred to minimal media with 2% glucose , and grown to log phase at 30 
  c . a digoxigenin - labeled 
  oligo(dt ) probe and fluorescein isothiocyanate - conjugated anti - digoxigenin 
  antibody ( 1:200 dilution , roche molecular biochemicals ) were used to localize 
  poly(a ) rna . for all samples , cells were also stained with 
  4,6-diamidino-2-phenylindole - dihydrochloride ( dapi ) to detect chromatin 
  and hence the position of the nucleus . to quantitate the percentage of cells processed for fish that showed 
  nuclear accumulation of poly(a ) rna , nab2 , nab2 f72d , nab2 f73d , mex67 - 5 
  nab2 , mex67 - 5 nab2 f72d , and mex67 - 5 nab2 f73d cells probed with 
  oligo(dt ) to visualize poly(a ) rna and 10 fields of cells for each strain were 
  imaged . for each field of cells , cells were counted and scored using imagej 
  version 1.4 software , and the percentage of cells with nuclear poly(a ) rna was 
  calculated . cells were scored as showing nuclear poly(a ) rna if the 
  fluorescent signal in the nucleus was greater than the signal in the 
  cytoplasm . the mean percentage of cells with nuclear poly(a ) rna was 
  calculated from the percentages in the 10 fields of cells . variation in the 
  percentage nuclear poly(a ) rna in each field from the mean was assessed by 
  standard deviation ( square root of the mean squared ) . all scoring of nuclear 
  poly(a ) rna was performed blind to avoid biasing results . in each field of 
  cells , at least 195 nab2 and nab2 mutant cells and at least 
  43 mex67 - 5 nab2 and mex67 - 5 nab2 mutant cells were counted . 
  the fold increase in nuclear poly(a ) rna accumulation in nab2 mutant 
  cells relative to that in nab2 or mex67 - 5 nab2 cells was 
  calculated by dividing the mean percentage of nuclear poly(a ) rna in 
  nab2 mutant cells by the mean percentage in corresponding 
  nab2 wild - type cells . cells expressing rgg - nab2-gfp or processed 
  for fish were visualized by using filters from chroma technology ( brattleboro , 
  vt ) and an olympus bx60 direct fluorescence microscope equipped with a 
  photometric quantix digital camera from roper scientific ( tucson , az ) . nab2 functional assay  in vivo functional analysis 
  of nab2 was carried out through a standard plasmid shuffle assay combined with 
  serial dilution and spotting or growth curves . to generate single mutants of 
  nab2 or double mutants of nab2 and mex67 - 5 or 
  yra18 , nab2 cells ( acy427 ) , nab2 
  mex67 - 5 cells ( nab2 mex67 cells 
  ( swy3601 ) containing mex67 - 5 trp1 plasmid ( prs314-mex67 - 5 ) ) 
  or nab2 yra18 cells ( nab2 
  yra1 cells ( fsy2327 ) containing yra18 trp1 
  plasmid ( pfs2152 ) ) containing nab2 ura3 maintenance plasmid ( pac636 ) 
  were transformed with nab2 ( pac717 ) , nab2 n 
  ( pac1152 ) , nab2 f72d ( pac2351 ) , or nab2 f73d ( pac2352 ) 
  leu2 plasmids and selected on leuura minimal 
  media with 2% glucose . cells were grown overnight at 30 c to saturation 
  in leuura minimal media with 2% glucose . cell 
  concentrations were normalized by a600 and cultures were 
  serially diluted in sterile h2o to obtain 10,000 , 1000 , 100 , 
  10 , or 1 cell per 3-l volume . these dilutions were spotted onto control 
  leuura minimal media , where the nab2 ura3 
  maintenance plasmid is maintained , or ura minimal media containing 
  5-fluoroorotic acid ( 5-foa ) , which selects for cells that have lost the 
  nab2 ura3 maintenance plasmid 
  ( 56 ) . growth of 
  nab2 , nab2 mex67 - 5 , or 
  nab2 yra18 cells harboring nab2 , nab2 
  n , nab2 f72d , or nab2 f73d as the sole copy of 
  nab2 was examined at 30 c . for growth curve analysis , 
  nab2 or nab2 mex67 - 5 cells harboring 
  nab2 , nab2 f72d , or nab2 f73d as the sole copy of 
  nab2 were grown overnight at 30 c to saturation in 
  leuura minimal media with 2% glucose . cell 
  concentrations were normalized by a600 , diluted 100-fold 
  in 100 l of leuura minimal media with 2% glucose 
  and added to wells of a microwell f96 microtiter plate ( nunc ) . cells in plate wells were grown at 30 c with shaking 
  and absorbance at a600 was measured every 30 min for 40 h 
  in an elx808 ultra microplate reader with kcjunior software 
  ( bio - tek instruments , inc . ) . duplicate sample absorbance for time points were 
  averaged and plotted using microsoft excel for mac 2004 ( microsoft corp . ) . identification of a minimal nab2-binding region in 
  mlp1previous work demonstrated that the globular , acidic 
  c - terminal domain of mlp1 ( ct - mlp1 , residues 14901875 ) interacts with 
  the hnrnp , nab2 , both in vitro and in vivo and that ectopic 
  expression of ct - mlp1 causes nuclear accumulation of poly(a ) rna 
  ( 38 ) . these data indicate that 
  mlp1 is involved in poly(a ) rna export and suggest that one function of nab2 
  is to serve as a molecular link between mlp1 and poly(a ) rna . to test this 
  model and gain further insight into the molecular basis for the nab2/mlp1 
  interaction , we employed a series of ct - mlp1 truncation mutants with 
  progressive deletions from the c - terminal end of mlp1 ( truncation mutants 
  ct15 , see fig . 1a ) to map the region within ct - mlp1 that interacts 
  directly with nab2 . these truncation mutants were used in both in 
  vitro and in vivo binding assays with nab2 . for in 
  vitro binding , recombinant gst , gst - ct - mlp1 , gst - ct2 , ct4 , or ct5 bound 
  to glutathione beads was incubated with soluble recombinant his - nab2 and the 
  bound and unbound fractions were analyzed by immunoblotting with anti - his 
  antibody to detect bound nab2 . the results were quantitated as described under 
   experimental procedures .  as shown in 
  fig . 1b , we found that 
  ct - mlp1 ct2 ( residues 14901779 ) binds to nab2 with similar affinity 
  ( 98% bound ) as intact ct - mlp1 ( residues 14901875 ) , whereas ct4 
  ( residues 14901682 ) binds more weakly to nab2 ( 16% bound ) compared with 
  ct - mlp1 , and ct5 ( residues 14901585 ) shows no binding to nab2 ( 1% 
  bound ) . as a control , gst alone does not bind to nab2 . this result suggests 
  that the nab2-binding region within ct - mlp1 lies within residues 
  14901779 . the observation that ct - mlp1 residues 14901779 ( ct2 ) binds to nab2 , 
  but residues 14901585 ( ct5 ) does not interact defines the c - terminal 
  end of the minimal nab2-binding region within ct - mlp1 . to further delineate 
  the n - terminal end of the minimal nab2-binding region , we generated two 
  additional ct - mlp1 truncation mutants , ct6 ( residues 15861779 ) and ct7 
  ( residues 16771779 ) ( fig . recombinant gst , gst - ct - mlp1 , gst - ct6 , or gst - ct7 
  bound to glutathione beads was incubated with soluble recombinant his - nab2 and 
  bound and unbound fractions were analyzed by immunoblotting with anti - his 
  antibody to detect bound nab2 . as shown in 
  fig . 1c , we found that 
  ct - mlp1 mutant ct6 ( residues 15861779 ) binds to nab2 to the same extent 
  ( > 100% bound ) as the intact ct - mlp1 ( residues 14901875 ) , whereas ct7 
  ( residues 16771779 ) does not bind to nab2 ( 2% bound ) . these data 
  indicate that the nab2-binding region within ct - mlp1 resides within residues 
  15861779 . figure 1.mapping of ct - mlp1 reveals a nab2-binding region between residues 1586 
  and 1779 . a , schematic depicting sizes of ct - mlp1 truncation 
  mutants ( ct17 ) and the nab2-binding domain of mlp1 
  ( mlp1-nbd ) constructed . b , ct - mlp1 truncation mutant ct2 
  ( residues 14901779 ) binds to nab2 in vitro , but mutant ct5 
  ( residues 14901585 ) does not bind to nab2 . recombinant gst , gst - ct - mlp1 
  ( ct - mlp1 ) , or the gst - ct - mlp1 truncation mutant ( ct2 , ct4 , 
  ct5 ) bound to glutathione - sepharose beads were incubated with recombinant 
  his - tagged nab2 ( nab2 ) and unbound ( u ) and bound 
  ( b ) fractions were analyzed by immunoblotting with anti - his antibody . the percentage of his - nab2 band intensity in each bound fraction relative to 
  the his - nab2 band intensity in the bound fraction of gst - ct - mlp1 is indicated 
  below the immunoblot . c , ct - mlp1 truncation mutant ct6 
  ( residues 15861779 ) binds to nab2 in vitro . recombinant gst , 
  gst - ct - mlp1 ( ct - mlp1 ) , or the gst - ct - mlp1 truncation mutant ( ct6 , 
  ct7 ) bound to glutathione - sepharose beads was incubated with recombinant 
  his - tagged nab2 ( nab2 ) and unbound ( u ) and bound 
  ( b ) fractions were analyzed by immunoblotting with anti - his antibody . the percentage of his - nab2 band intensity in each bound fraction relative to 
  the his - nab2 band intensity in the bound fraction of gst - ct - mlp1 is indicated 
  below the immunoblot . d , expression of ct - mlp1 truncation 
  mutant ct2 ( residues 14901779 ) causes relocalization of 
  rgg - nab2-gfp to the nucleus . ct - mlp1 and ct - mlp1 truncation mutants 
  ( ct15 ) were expressed in yeast cells expressing a nab2 mutant 
  fused to gfp ( rgg - nab2-gfp ) , which displays localization throughout the 
  cell ( 36 ) , and 
  rgg - nab2-gfp was visualized by direct fluorescence microscopy . mapping of ct - mlp1 reveals a nab2-binding region between residues 1586 
  and 1779 . a , schematic depicting sizes of ct - mlp1 truncation 
  mutants ( ct17 ) and the nab2-binding domain of mlp1 
  ( mlp1-nbd ) constructed . b , ct - mlp1 truncation mutant ct2 
  ( residues 14901779 ) binds to nab2 in vitro , but mutant ct5 
  ( residues 14901585 ) does not bind to nab2 . recombinant gst , gst - ct - mlp1 
  ( ct - mlp1 ) , or the gst - ct - mlp1 truncation mutant ( ct2 , ct4 , 
  ct5 ) bound to glutathione - sepharose beads were incubated with recombinant 
  his - tagged nab2 ( nab2 ) and unbound ( u ) and bound 
  ( b ) fractions were analyzed by immunoblotting with anti - his antibody . the percentage of his - nab2 band intensity in each bound fraction relative to 
  the his - nab2 band intensity in the bound fraction of gst - ct - mlp1 is indicated 
  below the immunoblot . c , ct - mlp1 truncation mutant ct6 
  ( residues 15861779 ) binds to nab2 in vitro . recombinant gst , 
  gst - ct - mlp1 ( ct - mlp1 ) , or the gst - ct - mlp1 truncation mutant ( ct6 , 
  ct7 ) bound to glutathione - sepharose beads was incubated with recombinant 
  his - tagged nab2 ( nab2 ) and unbound ( u ) and bound 
  ( b ) fractions were analyzed by immunoblotting with anti - his antibody . the percentage of his - nab2 band intensity in each bound fraction relative to 
  the his - nab2 band intensity in the bound fraction of gst - ct - mlp1 is indicated 
  below the immunoblot . d , expression of ct - mlp1 truncation 
  mutant ct2 ( residues 14901779 ) causes relocalization of 
  rgg - nab2-gfp to the nucleus . ct - mlp1 and ct - mlp1 truncation mutants 
  ( ct15 ) were expressed in yeast cells expressing a nab2 mutant 
  fused to gfp ( rgg - nab2-gfp ) , which displays localization throughout the 
  cell ( 36 ) , and 
  rgg - nab2-gfp was visualized by direct fluorescence microscopy . we used a secondary in vivo nab2 binding assay to confirm the 
  in vitro data mapping the nab2-binding region of mlp1 to residues 
  15861779 . previously , we developed a rgg - nab2 relocalization 
  assay that reports on the interaction between nab2 and mlp1 in vivo 
  ( 38 ) . in contrast to wild - type 
  nab2 , which shows nuclear localization at steady - state 
  ( 35 ) , rgg - nab2 shows 
  diffuse nuclear and cytoplasmic localization at steady - state due to lack of 
  the rgg domain ( 36 ) , which is 
  required for interaction with the nab2 import receptor kap104 
  ( 37 , 
  57 ) . expression of ct - mlp1 , 
  which localizes to the nucleus 
  ( 38 ) , causes nuclear 
  relocalization of rgg - nab2 fused to gfp ( rgg - nab2-gfp ) , which 
  normally locates throughout the yeast cell 
  ( 36 ) . ct - mlp1-induced 
  relocalization of rgg - nab2-gfp to the nucleus is consistent with 
  ct - mlp1 binding to nab2 in vivo . therefore , to confirm the in 
  vitro binding data using this in vivo binding assay , we 
  expressed ct - mlp1 and ct - mlp1 truncation mutants ct15 in yeast from a 
  galactose - inducible gal110 promoter . to ensure nuclear 
  targeting of each of the ct - mlp1 deletions , the ct15 sequences were 
  fused to an engineered bipartite nuclear localization signal based on the 
  simian virus 40 ( sv40 ) nuclear localization signal 
  ( 58 ) . wild - type yeast cells 
  expressing rgg - nab2-gfp were induced to express ct - mlp1 and ct15 
  by the addition of galactose and the localization of rgg - nab2-gfp was 
  assessed by direct fluorescence microscopy . the expression and localization of 
  ct - mlp1 and ct15 were confirmed by immunoblotting and gfp fusion 
  localization ( data not shown ) . as shown in 
  fig . 1d , ct - mlp1 
  truncation mutant ct1 ( residues 14901811 ) and ct2 ( residues 
  14901779 ) relocalize rgg - nab2-gfp to the nucleus to a similar 
  degree as the intact ct - mlp1 ( residues 14901875 ) . truncation mutants 
  ct3 ( residues 14901717 ) and ct4 ( residues 14901682 ) partially 
  relocalize rgg - nab2-gfp to the nucleus compared with ct - mlp1 and 
  truncation mutant ct5 ( residues 14901585 ) does not relocalize 
  rgg - nab2-gfp to the nucleus . this result indicates that ct - mlp1 
  residues 14901779 binds to nab2 , whereas ct - mlp1 residues 
  14901585 do not interact with nab2 in vivo . taken together , 
  the nab2 binding assays strongly suggest the nab2-binding region within 
  ct - mlp1 maps to residues 15861779 . the nab2-binding region of mlp1 causes nuclear accumulation of poly(a ) 
  rna  expression of ct - mlp1 ( residues 14901875 ) causes nuclear 
  accumulation of poly(a ) rna 
  ( 38 ) . to map the region within 
  ct - mlp1 that causes poly(a ) rna accumulation and assess whether it corresponds 
  to the nab2-binding region , we expressed ct - mlp1 and ct - mlp1 truncation 
  mutants ct15 in yeast from a galactose - inducible promoter . wild - type 
  yeast cells were induced to express ct - mlp1 and ct15 by the addition of 
  galactose and samples were processed for fish using an oligo(dt ) probe to 
  visualize bulk poly(a ) rna . ct - mlp1 truncation mutants ct1 ( residues 
  14901811 ) and ct2 ( residues 14901779 ) cause nuclear accumulation 
  of poly(a ) rna to a similar extent as the intact ct - mlp1 ( residues 
  14901875 ) , ct3 ( residues 14901717 ) causes partial nuclear 
  accumulation of poly(a ) rna compared with ct - mlp1 , and ct4 ( residues 
  14901682 ) and ct5 ( residues 14901585 ) do not cause poly(a ) rna 
  accumulation in the nucleus ( fig . 
  2 ) . this result indicates that the region within ct - mlp1 that is 
  sufficient to cause nuclear accumulation of poly(a ) rna maps to residues 
  14901779 . thus , both the nab2-binding and poly(a ) rna interacting 
  regions of ct - mlp1 reside within residues 14901779 . delineation of the mlp1-nab2 binding domain ( mlp1-nbd)as a 
  complement to our mapping using engineered deletions , we also isolated a 
  proteolytic fragment of the intact ct - mlp1 that bound to nab2 in 
  vitro and identified the mlp1 tryptic peptides within this fragment by 
  mass spectroscopy ( see supplemental fig . s1 and supplemental table 1 ) . we 
  mapped these peptides within ct - mlp1 and used the information to refine the 
  c - terminal end of the nab2-binding region of mlp1 to residue 1768 . we termed 
  the final nab2-binding region of mlp1 : mlp1-nab2-binding domain ( mlp1-nbd ; 
  residues 15861768 ) ( fig . figure 2.mapping of ct - mlp1 defines a region between residues 1490 and 1779 
  sufficient to cause nuclear poly(a ) rna accumulation . ct - mlp1 and ct - mlp1 
  truncation mutants ( ct15 ) were expressed in yeast cells and 
  poly(a ) rna ( poly(a ) ) was visualized by fluorescence in 
  situ hybridization with an oligo(dt ) probe as described under 
   experimental procedures . mapping of ct - mlp1 defines a region between residues 1490 and 1779 
  sufficient to cause nuclear poly(a ) rna accumulation . ct - mlp1 and ct - mlp1 
  truncation mutants ( ct15 ) were expressed in yeast cells and 
  poly(a ) rna ( poly(a ) ) was visualized by fluorescence in 
  situ hybridization with an oligo(dt ) probe as described under 
   experimental procedures . cells were stained with dapi to 
  visualize the position of the nucleus . corresponding differential interference 
  contrast ( dic ) images are shown . the mlp1-nbd interacts with the nab2 n - terminal 
  domain  recent domain analysis of the hnrnp nab2 has established 
  that the n - terminal domain of nab2 ( nab2-n , residues 197 ) binds to both 
  ct - mlp1 and full - length mlp1 in vitro 
  ( 49 ) . to determine whether the 
  nab2-binding domain of mlp1 ( mlp1-nbd , residues 15861768 ) interacts 
  directly with nab2-n , we analyzed the interaction by an in vitro 
  binding assay using purified recombinant proteins . a control protein , 
  ovalbumin , or recombinant his - nab2-n - wt ( residues 197 ) coupled to 
  sepharose beads was incubated with soluble recombinant his - mlp1-nbd and bound 
  fractions were analyzed by sds - page and coomassie staining . as shown in 
  fig . 3a , mlp1-nbd 
  binds to nab2-n - wt , but does not bind to control ovalbumin beads . to further 
  support this direct interaction and test its specificity , we tested whether 
  mlp1-nbd can be co - purified with nab2-n from a complex protein mixture . we 
  combined cell lysates from e. coli expressing his - mlp1-nbd and e. 
  coli expressing gst - nab2-n , incubated the mixed lysates with glutathione 
  beads and analyzed the bound fraction by sds - page and coomassie staining . we 
  find that mlp1-nbd does indeed co - purify with nab2-n from a complex mixture of 
  bacterial proteins , highlighting the specificity of this interaction 
  ( fig . 3b ) . together , 
  the experiments demonstrate that the nab2-binding domain of mlp1 ( residues 
  15861768 ) interacts directly with the n - terminal domain of nab2 
  ( residues 197 ) . importantly , previous studies show that the interaction 
  between ct - mlp1 and nab2 in yeast lysate is insensitive to rnase treatment and 
  therefore rna - independent and also that nab2-n does not interact with poly(a ) 
  rna in vitro ( 38 , 
  49 ) . the direct interaction 
  between nab2-n and mlp1-nbd is thus most likely rna - independent as well . figure 3.the nab2-binding domain of mlp1 directly interacts with nab2 and causes 
  nuclear accumulation of poly(a ) rna . a , the nab2-binding domain 
  of mlp1 ( mlp1-nbd ; residues 15861768 ) binds to the n - terminal domain of 
  nab2 ( nab2-n ; residues 197 ) in vitro . his - nab2-n ( nab2-n ) or , 
  as a control , ovalbumin ( oval . ) coupled to sepharose beads was 
  incubated with his - mlp1-nbd and bound fractions were analyzed by sds - page and 
  coomassie staining . b , the nab2-binding domain of mlp1 ( mlp1-nbd ) 
  co - purifies with the n - terminal domain of nab2 ( nab2-n ) . cell lysates from 
  e. coli expressing his - mlp1-nbd and e. coli expressing 
  gst - nab2-n were combined , incubated with glutathione beads , and beads were 
  washed three times . input combined lysate ( i ) , unbound ( u ) 
  fraction , three wash fractions ( w1 , w2 , w3 ) , and bound ( b ) 
  fraction were analyzed by sds - page and coomassie staining . c , 
  expression of the nab2-binding domain of mlp1 ( mlp1-nbd ) relocalizes 
  rgg - nab2-gfp to the nucleus . ct - mlp1 and mlp1-nbd were expressed in 
  yeast cells expressing rgg - nab2-gfp , which displays localization 
  throughout the cell ( 36 ) , and 
  rgg - nab2-gfp was visualized by direct fluorescence microscopy . d , expression 
  of the nab2-binding domain of mlp1 ( mlp1-nbd ) causes nuclear accumulation of 
  poly(a ) rna . ct - mlp1 and mlp1-nbd were expressed in yeast cells and poly(a ) 
  rna ( poly(a ) ) was visualized by fluorescence in 
  situ hybridization with an oligo(dt ) probe as described under 
   experimental procedures . the nab2-binding domain of mlp1 directly interacts with nab2 and causes 
  nuclear accumulation of poly(a ) rna . a , the nab2-binding domain 
  of mlp1 ( mlp1-nbd ; residues 15861768 ) binds to the n - terminal domain of 
  nab2 ( nab2-n ; residues 197 ) in vitro . his - nab2-n ( nab2-n ) or , 
  as a control , ovalbumin ( oval . ) coupled to sepharose beads was 
  incubated with his - mlp1-nbd and bound fractions were analyzed by sds - page and 
  coomassie staining . b , the nab2-binding domain of mlp1 ( mlp1-nbd ) 
  co - purifies with the n - terminal domain of nab2 ( nab2-n ) . cell lysates from 
  e. coli expressing his - mlp1-nbd and e. coli expressing 
  gst - nab2-n were combined , incubated with glutathione beads , and beads were 
  washed three times . input combined lysate ( i ) , unbound ( u ) 
  fraction , three wash fractions ( w1 , w2 , w3 ) , and bound ( b ) 
  fraction were analyzed by sds - page and coomassie staining . c , 
  expression of the nab2-binding domain of mlp1 ( mlp1-nbd ) relocalizes 
  rgg - nab2-gfp to the nucleus . ct - mlp1 and mlp1-nbd were expressed in 
  yeast cells expressing rgg - nab2-gfp , which displays localization 
  throughout the cell ( 36 ) , and 
  rgg - nab2-gfp was visualized by direct fluorescence microscopy . d , expression 
  of the nab2-binding domain of mlp1 ( mlp1-nbd ) causes nuclear accumulation of 
  poly(a ) rna . ct - mlp1 and mlp1-nbd were expressed in yeast cells and poly(a ) 
  rna ( poly(a ) ) was visualized by fluorescence in 
  situ hybridization with an oligo(dt ) probe as described under 
   experimental procedures . corresponding differential interference 
  contrast images are shown . to further compare the mlp1-nbd ( residues 15861768 ) with the intact 
  ct - mlp1 , we asked whether the mlp1-nbd can both interact with nab2 and cause 
  nuclear accumulation of poly(a ) rna in vivo . in the 
  rgg - nab2-gfp relocalization assay , we expressed nuclear - targeted 
  ct - mlp1 and mlp1-nbd from a galactose - inducible promoter and found that 
  mlp1-nbd ( residues 15861768 ) relocalizes most rgg - nab2-gfp to 
  the nucleus , although not quite to the same extent as ct - mlp1 ( residues 
  14901875 ) ( fig . if the expression of mlp1-nbd affects 
  poly(a ) rna localization , we expressed mlp1-nbd and ct - mlp1 in yeast cells and 
  localized the poly(a ) rna by fish with an oligo d(t ) probe . we find that the 
  mlp1-nbd does induce accumulation of poly(a ) rna within the nucleus , although 
  not quite to the same degree as ct - mlp1 
  ( fig . these 
  results indicate that the mlp1-nbd ( residues 15861768 ) within ct - mlp1 
  is primarily responsible for ct - mlp1-induced nuclear poly(a ) rna accumulation , 
  but that small contributions are also made by other residues outside this 
  region of ct - mlp1 . overall , these data confirm that the mlp1-nbd , like the 
  intact ct - mlp1 , has the capacity both to bind nab2 in vivo and to 
  impact poly(a ) rna localization . to measure quantitatively the interaction between the mlp1-nbd and nab2-n , 
  we examined the binding of s - tag - mlp1-nbd to gst - nab2-n wild - type ( wt ) at 
  increasing concentrations of s - tag - mlp1-nbd in a solid - phase microtiter plate 
  binding assay ( 54 ) . this 
  analysis generated a saturation binding curve and an apparent dissociation 
  constant ( kd ) value for mlp1-nbd binding to nab2-n - wt . we 
  find that mlp1-nbd binds to wild - type nab2-n with an apparent 
  kd of 1.3  0.4 m 
  ( fig . 4b , 
  table 2 ) , indicating that the 
  relative binding affinity between mlp1-nbd and nab2-n is in the micromolar 
  range . the rather weak affinity between mlp1 and nab2 likely specific amino acid substitutions disrupt the nab2-n / mlp1-nbd 
  interaction  the crystal structure of the n - terminal domain of nab2 
  ( nab2-n , residues 197 ) shows that this domain is based on a five 
  -helix bundle with a pwi - like fold 
  ( 49 ) . a nab2 hydrophobic patch 
  centered on a phenylalanine residue , phe 
  ( fig . 4a ) , was 
  proposed to be an important constituent of the nab2/mlp1 interaction interface 
  ( 49 ) . consistent with this 
  hypothesis , we showed that recombinant nab2-n - f73d does not bind to mlp1 , 
  whereas nab2-n - f72d is still competent to bind mlp1 , in an in vitro 
  bead binding assay . to assess the impact of amino acid substitutions at nab2 
  phe or phe on the relative binding affinity of nab2 
  for mlp1 , we measured the binding of s - tag - mlp1-nbd to gst - nab2-n mutants 
  f72d , f73d , or f73w at increasing concentrations of s - tag - mlp1-nbd in the 
  solid - phase binding assay . we find that the interaction between nab2-n - f73d 
  and mlp1-nbd is undetectable in this assay , whereas mlp1-nbd binds to 
  nab2-n - f72d with roughly 2-fold lower relative affinity 
  ( kd  2.3  1.1 m ) compared with 
  wild - type ( fig . furthermore , we find 
  that mlp1-nbd binds to nab2-n - f73w with 1.5-fold higher affinity 
  ( kd  0.8  0.2 m ) compared with 
  wild - type nab2-n ( fig . 
  4b , table 
  2 ) . the data reveal that substituting nab2-n residue 
  phe with asp significantly reduces the relative binding affinity 
  between nab2-n and the mlp1-nbd , supporting the idea that nab2 
  phe is critical for interaction with mlp1 . the impaired binding 
  of nab2-n to mlp1-nbd seen with the hydrophilic substitution , f73d , together 
  with the enhanced binding seen with the hydrophobic substitution , f73w , is 
  consistent with the proposal 
  ( 49 ) that hydrophobic 
  interactions contribute significantly to the nab2/mlp1 interface . figure 4.the nab2-binding domain of mlp1 binds to the n - terminal domain of nab2 
  with micromolar affinity and nab2 f73d substitution disrupts the nab2/mlp1 
  interaction . a , surface representation of the n - terminal domain 
  of nab2 ( nab2-n ) showing the putative mlp1 interaction site ( gray ) 
  identified by grant et al . phe 
  ( dark gray ) is central to this site and its hydrophobic aromatic side 
  chain is completely exposed to the solvent . b , the binding affinity 
  of the nab2-binding domain of mlp1 ( mlp1-nbd ; residues 15861768 ) for 
  nab2-n wild - type and mutants f72d , f73d , and f73w ( residues 197 ) was 
  measured quantitatively in a solid - phase binding assay 
  ( 54 ) . binding of 
  s - tag - mlp1-nbd ( mlp1-nbd ) to gst - nab2-n wild - type ( nab2-n - wt ) , 
  gst - nab2-n - f72d ( nab2-n - f72d ) , gst - nab2-n - f73d 
  ( nab2-n - f73d ) , or gst - nab2-n - f73w ( nab2-n - f73w ) at 
  increasing concentrations of s - tag - mlp1-nbd was measured at optical density of 
  450 nm ( a450 ) as described under  experimental 
  procedures .  measurements at a450 ( amount bound ) 
  were used to generate saturation binding curves by non - linear regression . standard error bars for a450 readings from 
  triplicate samples of gst - nab2-n - wt and mutants at each s - tag - mlp1-nbd 
  concentration are indicated . these binding curves were used to calculate 
  apparent dissociation constants for binding between mlp1-nbd and nab2-n 
  wild - type ( kd  1.3  0.4 m ) , 
  nab2-n - f72d ( kd  2.3  1.1 m ) , 
  or nab2-n - f73w ( kd  0.8  0.2 
  m ) ( see table 
  2 ) . the weak binding between mlp1-nbd and nab2-n - f73d was 
  indistinguishable from nonspecific binding , indicating that the f73d 
  substitution essentially disrupts the nab2/mlp1 interaction . the nab2-binding domain of mlp1 binds to the n - terminal domain of nab2 
  with micromolar affinity and nab2 f73d substitution disrupts the nab2/mlp1 
  interaction . a , surface representation of the n - terminal domain 
  of nab2 ( nab2-n ) showing the putative mlp1 interaction site ( gray ) 
  identified by grant et al . phe 
  ( dark gray ) is central to this site and its hydrophobic aromatic side 
  chain is completely exposed to the solvent . b , the binding affinity 
  of the nab2-binding domain of mlp1 ( mlp1-nbd ; residues 15861768 ) for 
  nab2-n wild - type and mutants f72d , f73d , and f73w ( residues 197 ) was 
  measured quantitatively in a solid - phase binding assay 
  ( 54 ) . binding of 
  s - tag - mlp1-nbd ( mlp1-nbd ) to gst - nab2-n wild - type ( nab2-n - wt ) , 
  gst - nab2-n - f72d ( nab2-n - f72d ) , gst - nab2-n - f73d 
  ( nab2-n - f73d ) , or gst - nab2-n - f73w ( nab2-n - f73w ) at 
  increasing concentrations of s - tag - mlp1-nbd was measured at optical density of 
  450 nm ( a450 ) as described under  experimental 
  procedures .  measurements at a450 ( amount bound ) 
  were used to generate saturation binding curves by non - linear regression . standard error bars for a450 readings from 
  triplicate samples of gst - nab2-n - wt and mutants at each s - tag - mlp1-nbd 
  concentration are indicated . these binding curves were used to calculate 
  apparent dissociation constants for binding between mlp1-nbd and nab2-n 
  wild - type ( kd  1.3  0.4 m ) , 
  nab2-n - f72d ( kd  2.3  1.1 m ) , 
  or nab2-n - f73w ( kd  0.8  0.2 
  m ) ( see table 
  2 ) . the weak binding between mlp1-nbd and nab2-n - f73d was 
  indistinguishable from nonspecific binding , indicating that the f73d 
  substitution essentially disrupts the nab2/mlp1 interaction . functional significance of the nab2/mlp1 interaction  with 
  the knowledge gained from the nab2-n structure and engineered nab2 mutants , we 
  proceeded to assess the functional consequence of disrupting the nab2/mlp1 
  interaction in vivo and define the way in which this interaction 
  contributes to mrna export . as the nab2 gene is essential for 
  viability ( 35 ) , we examined 
  whether nab2-f73d or nab2-f72d ( as a control ) could replace wild - type nab2 in 
  yeast cells . as a further control in these assays , we used a previously 
  characterized nab2 mutant , n , lacking the nab2 n - terminal domain 
  altogether ( residues 497 ) , which confers a severe slow growth phenotype 
  when expressed as the only cellular copy of nab2 
  ( 36 ) . using a plasmid shuffle 
  assay , nab2 yeast cells maintained by a nab2 ura3 
  plasmid and also containing nab2 , nab2 n , f72d or 
  f73d leu2 test plasmids were grown to saturation , serially diluted in 
  10-fold dilutions , and spotted on control plates ( where the nab2 ura3 
  maintenance plasmid is retained ) and plates containing 5-foa to remove the 
  nab2 ura3 maintenance plasmid and leave the test plasmids as the sole 
  cellular copies of nab2 . both nab2 f72d and nab2 f73d cells 
  grow similarly to wild - type nab2 cells on 5-foa plates 
  ( fig . however , as 
  anticipated , nab2 n cells show a slow growth 
  phenotype . we confirmed that nab2 f72d and nab2 f73d cells 
  grow similarly to wild - type nab2 cells by measuring the optical 
  density of the cells in liquid culture over time and generating quantitative 
  growth curves for the cells ( fig . this result indicates that substituting nab2 residue 
  phe with asp and disrupting the nab2/mlp1 interaction does not 
  significantly affect cell growth in a wild - type background . additionally , 
  given the knowledge that the n - terminal domain of nab2 is required for proper 
  nab2 function ( including binding to other proteins such as gfd1 
  ( 49 , 
  51 ) ) , the data suggest that 
  the f73d substitution does not severely impact the overall structure of the 
  nab2 protein . figure 5.nab2 f73d mutant cells grow similarly to wild - type cells but 
  display nuclear accumulation of poly(a ) rna . a , nab2 f73d cells 
  are viable and grow similarly to wild - type nab2 cells . nab2 cells maintained by a nab2 ura3 plasmid and 
  containing nab2 , nab2 n , nab2 f72d , or nab2 f73d 
  leu2 test plasmids were grown to saturation , serially diluted in 10-fold 
  dilutions , and spotted on control and 5-foa plates . b , growth curve analysis of nab2 f73d cells confirms 
  that they grow similarly to wild - type nab2 cells . f73d plasmids were grown to saturation , diluted , and their optical 
  density was measured at a600 for 40 h as described under 
   experimental procedures . f73d 
  plasmids were grown in liquid culture at 30 c and poly(a ) rna 
  ( poly(a ) ) was visualized by fish using an oligo(dt ) probe as 
  described under cells were stained with 
  dapi to visualize the position of the nucleus . corresponding differential 
  interference contrast ( dic ) images are shown . experimental procedures  to assess 
  the mean percentage of cells with nuclear accumulation of poly(a ) rna within 
  10 fields of at least 195 cells each ( indicated to the right of dic images ) . variation in the percentage of cells with nuclear poly(a ) rna in each field of 
  cells from the mean was assessed by calculating the standard deviation : 
  nab2 , 3.2  1.0% ; nab2 f72d , 3.9  1.5% ; and 
  nab2 f73d , 8.2  2.0% . thus , nab2 f73d cells exhibit 
  a 2.5-fold increase in nuclear poly(a ) rna accumulation relative to 
  nab2 cells , whereas nab2 f72d cells show only a modest 
  1.2-fold increase . the percentages of nuclear poly(a ) rna accumulation 
  observed in nab2 f73d cells were typical of multiple independent 
  experiments . nab2 f73d mutant cells grow similarly to wild - type cells but 
  display nuclear accumulation of poly(a ) rna . a , nab2 f73d cells 
  are viable and grow similarly to wild - type nab2 cells . nab2 cells maintained by a nab2 ura3 plasmid and 
  containing nab2 , nab2 n , nab2 f72d , or nab2 f73d 
  leu2 test plasmids were grown to saturation , serially diluted in 10-fold 
  dilutions , and spotted on control and 5-foa plates . b , growth curve analysis of nab2 f73d cells confirms 
  that they grow similarly to wild - type nab2 cells . f73d plasmids were grown to saturation , diluted , and their optical 
  density was measured at a600 for 40 h as described under 
   experimental procedures . f73d 
  plasmids were grown in liquid culture at 30 c and poly(a ) rna 
  ( poly(a ) ) was visualized by fish using an oligo(dt ) probe as 
  described under  experimental procedures . cells were stained with 
  dapi to visualize the position of the nucleus . corresponding differential 
  interference contrast ( dic ) images are shown . experimental procedures  to assess 
  the mean percentage of cells with nuclear accumulation of poly(a ) rna within 
  10 fields of at least 195 cells each ( indicated to the right of dic images ) . variation in the percentage of cells with nuclear poly(a ) rna in each field of 
  cells from the mean was assessed by calculating the standard deviation : 
  nab2 , 3.2  1.0% ; nab2 f72d , 3.9  1.5% ; and 
  nab2 f73d , 8.2  2.0% . thus , nab2 f73d cells exhibit 
  a 2.5-fold increase in nuclear poly(a ) rna accumulation relative to 
  nab2 cells , whereas nab2 f72d cells show only a modest 
  1.2-fold increase . the percentages of nuclear poly(a ) rna accumulation 
  observed in nab2 f73d cells were typical of multiple independent 
  experiments . to determine whether disruption of the nab2/mlp1 interaction impacts mrna 
  export , we next assessed whether nab2 f72d or nab2 f73d 
  cells show nuclear accumulation of poly(a ) rna in vivo . we examined 
  poly(a ) rna localization in wild - type nab2 , nab2 f72d , and nab2 
  f73d cells by fish and then quantitated the percentage of cells with 
  nuclear poly(a ) rna accumulation as described under  experimental 
  procedures .  as a control , we also examined poly(a ) rna localization in 
  nab2 n cells , which show nuclear accumulation of 
  poly(a ) rna ( 36 ) . nab2 cells expressing nab2 , nab2 n , 
  f72d , or f73d were processed for fish using an oligo(dt ) probe 
  to visualize bulk poly(a ) rna . the nab2 f73d cells show a 2.5-fold 
  increase in nuclear accumulation of poly(a ) rna compared with wild - type cells , 
  whereas nab2 f72d cells show accumulation of poly(a ) rna in the 
  nucleus comparable with wild - type cells 
  ( fig . 5c ) . control 
  nab2 n cells show significant nuclear accumulation of 
  poly(a ) rna as expected ( 36 ) . this result suggests that the nab2/mlp1 interaction facilitates but is not 
  absolutely required for mrna export . because multiple mrna export factors contribute to efficient mrna export , 
  we considered the possibility that the interaction between nab2 and mlp1 might 
  contribute to a step in mrna export that is not normally rate - limiting in a 
  wild - type background . thus , an effect on overall cellular function as assayed 
  by cell growth might only be evident in a genetic background where additional 
  steps in mrna export are compromised or suboptimal . we therefore tested for 
  genetic interactions between nab2 f72d or nab2 f73d and mrna 
  export mutants , mex67 - 5 
  ( 8) or yra18 
  ( 21 ) , at the permissive growth 
  temperature for these conditional mutants . using the plasmid shuffle assay , 
  nab2 mex67 - 5 yeast cells harboring the nab2 
  ura3 maintenance plasmid and containing nab2 , nab2 n , 
  f72d or f73d leu2 test plasmids were grown to saturation , 
  serially diluted , and spotted on control and 5-foa plates . the mex67 - 5 
  nab2 f73d double mutant cells show a slow growth phenotype compared with 
  mex67 - 5 nab2 cells , whereas mex67 - 5 nab2 f72d cells show 
  growth similar to mex67 - 5 nab2 cells 
  ( fig . 6a ) . further 
  supporting a functional link between mex67 and nab2 , mex67 - 5 we confirmed the slow growth phenotype 
  of mex67 - 5 nab2 f73d cells relative to mex67 - 5 nab2 cells by 
  measuring the optical density of the cells in liquid cultures and generating 
  quantitative growth curves ( fig . nab2 
  yra18 yeast cells harboring the nab2 ura3 maintenance 
  plasmid and containing nab2 , nab2 n , f72d or f73d 
  leu2 test plasmids were grown to saturation and spotted on control and 
  5-foa plates . the yra18 nab2 f73d double mutant cells show a 
  slow growth phenotype compared with yra18 nab2 cells 
  ( fig . 6a ) , whereas 
  yra18 nab2 f72d cells show growth similar to yra18 
  nab2 cells . these results demonstrate that nab2 f73d genetically 
  interacts with mrna export mutants mex67 - 5 and yra18 at their 
  permissive temperatures . together , the data suggest that although disrupting 
  the nab2 interaction with mlp1 does not detectably impact yeast cell growth 
  when the mrna export pathway is fully functional , it does impact cell growth 
  when mrna export is suboptimal . figure 6.the nab2 f73d mutant displays genetic interactions with mrna export 
  mutants mex67 - 5 and yra18 and mex67 - 5 nab2 f73d cells show nuclear 
  poly(a ) rna accumulation . a , mex67 - 5 nab2 f73d cells or 
  yra18 nab2 f73d cells have a slow growth phenotype . nab2 mex67 - 5 or nab2 
  yra18 cells maintained by a nab2 ura3 plasmid and 
  containing nab2 , nab2 n , nab2 f72d , or nab2 f73d 
  leu2 test plasmids were grown to saturation , serially diluted in 10-fold 
  dilutions and spotted on control and 5-foa plates . b , growth curve analysis of mex67 - 5 nab2 f73d cells 
  confirms that they grow more slowly than mex67 - 5 nab2 cells . mex67 - 5 nab2 cells carrying only nab2 , nab2 f72d , or 
  nab2 f73d plasmids were grown to saturation , diluted , and their 
  optical density was measured at a600 for 40 h as described 
  under  experimental procedures . c , mex67 - 5 nab2 f73d 
  cells show nuclear accumulation of poly(a ) rna . nab2 
  mex67 - 5 cells carrying only nab2 , nab2 f72d , or nab2 
  f73d plasmids were grown in liquid culture at 30 c and poly(a ) rna 
  ( poly(a ) ) was visualized by fish using an oligo(dt ) probe as 
  described under cells were stained with 
  dapi to visualize the position of the nucleus . corresponding differential 
  interference contrast ( dic ) images are shown . experimental procedures  to assess 
  the mean percentage of cells with nuclear accumulation of poly(a ) rna within 
  10 fields of at least 43 cells each ( indicated to the right of the 
  dic images ) . variation in the percentage of cells with nuclear poly(a ) rna in 
  each field of cells from the mean was assessed by calculating the standard 
  deviation : mex67 - 5 nab2 , 18.6  4.8% ; mex67 - 5 nab2 
  f72d , 20.3  6.0% ; and mex67 - 5 nab2 f73d , 30.8  
  6.9 . thus , mex67 - 5 nab2 f73d cells exhibit a 1.7-fold increase in 
  nuclear poly(a ) rna accumulation relative to mex67 - 5 nab2 cells , 
  whereas mex67 - 5 nab2 f72d cells show only a slight 1.1-fold increase . the percentages of nuclear poly(a ) rna accumulation observed in mex67 - 5 
  nab2 f73d cells were typical of multiple independent experiments . the nab2 f73d mutant displays genetic interactions with mrna export 
  mutants mex67 - 5 and yra18 and mex67 - 5 nab2 f73d cells show nuclear 
  poly(a ) rna accumulation . a , mex67 - 5 nab2 f73d cells or 
  yra18 nab2 f73d cells have a slow growth phenotype . nab2 mex67 - 5 or nab2 
  yra18 cells maintained by a nab2 ura3 plasmid and 
  containing nab2 , nab2 n , nab2 f72d , or nab2 f73d 
  leu2 test plasmids were grown to saturation , serially diluted in 10-fold 
  dilutions and spotted on control and 5-foa plates . b , growth curve analysis of mex67 - 5 nab2 f73d cells 
  confirms that they grow more slowly than mex67 - 5 nab2 cells . mex67 - 5 nab2 cells carrying only nab2 , nab2 f72d , or 
  nab2 f73d plasmids were grown to saturation , diluted , and their 
  optical density was measured at a600 for 40 h as described 
  under  experimental procedures . c , mex67 - 5 nab2 f73d 
  cells show nuclear accumulation of poly(a ) rna . nab2 
  mex67 - 5 cells carrying only nab2 , nab2 f72d , or nab2 
  f73d plasmids were grown in liquid culture at 30 c and poly(a ) rna 
  ( poly(a ) ) was visualized by fish using an oligo(dt ) probe as 
  described under cells were stained with 
  dapi to visualize the position of the nucleus . corresponding differential 
  interference contrast ( dic ) images are shown . experimental procedures  to assess 
  the mean percentage of cells with nuclear accumulation of poly(a ) rna within 
  10 fields of at least 43 cells each ( indicated to the right of the 
  dic images ) . variation in the percentage of cells with nuclear poly(a ) rna in 
  each field of cells from the mean was assessed by calculating the standard 
  deviation : mex67 - 5 nab2 , 18.6  4.8% ; mex67 - 5 nab2 
  f72d , 20.3  6.0% ; and mex67 - 5 nab2 f73d , 30.8  
  6.9 . thus , mex67 - 5 nab2 f73d cells exhibit a 1.7-fold increase in 
  nuclear poly(a ) rna accumulation relative to mex67 - 5 nab2 cells , 
  whereas mex67 - 5 nab2 f72d cells show only a slight 1.1-fold increase . the percentages of nuclear poly(a ) rna accumulation observed in mex67 - 5 
  nab2 f73d cells were typical of multiple independent experiments . given that the nab2 f73d cells showed a mild mrna export defect in 
  a wild - type background , we wished to assess whether cells expressing nab2 f73d 
  in a mex67 - 5 mrna export mutant background , which showed a severe 
  cell growth defect , exhibit a similar or more pronounced mrna export defect . 
  to address this possibility , we examined poly(a ) rna localization in the 
  mex67 - 5 nab2 f73d cells and quantitated the percentage of cells with 
  nuclear poly(a ) rna accumulation . importantly , we examined the cells at the 
  permissive temperature of 30 c where mex67 - 5 cells show minimal 
  nuclear accumulation of poly(a ) rna 
  ( 8) . f73d were processed for fish with an oligo(dt ) probe to visualize bulk 
  poly(a ) rna . the mex67 - 5 nab2 f73d cells show a 1.7-fold increase in 
  nuclear accumulation of poly(a ) rna compared with mex67 - 5 nab2 cells 
  ( fig . the 
  combined results suggest that nab2-f73d , which disrupts the nab2/mlp1 
  interaction , impacts mrna export in vivo . in this study , we have examined the functional significance of the 
  interaction between the poly(a ) rna - binding protein , nab2 , and the nuclear 
  pore - associated protein , mlp1 , to test a model where nab2 facilitates 
  targeting of mrna for export via interaction with mlp1 . we find that 
  disruption of the nab2/mlp1 interaction in vivo causes nuclear 
  accumulation of poly(a ) rna . furthermore , the nab2 f73d mutant shows 
  genetic interactions with two genes essential for mrna export , mex67 
  and yra1 , supporting the hypothesis that nab2 binding to mlp1 
  contributes to efficient mrna export . in addition , we have characterized the 
  nab2/mlp1 interaction in detail and find that the nab2-binding domain of mlp1 
  ( mlp1-nbd ) is sufficient to bind to the n - terminal domain of nab2 ( nab2-n ) and 
  cause nuclear accumulation of poly(a ) rna , suggesting that nab2 is an 
  important molecular link between mlp1 and poly(a ) rna . we also find that 
  mlp1-nbd binds to nab2-n with micromolar affinity , indicating that the 
  nab2/mlp1 interaction , like other mrna export factor - nucleoporin interactions 
  ( 59 , 
  60 ) , is comparatively weak . together , the results are consistent with the notion that nab2 facilitates 
  targeting of mrna for export via transient interaction with mlp1 and acts in 
  concert with other mrna export factors to coordinate proper mrna export . we have mapped the nab2-binding domain of mlp1 ( mlp1-nbd ) to a 183-amino 
  acid region ( residues 15861768 ) within ct - mlp1 . analysis of the 
  mlp1-nbd protein sequence using phyre 
  ( www.sbg.bio.ic.ac.uk/phyre/html/index.html ) 
  suggests that it forms a predominantly -helical secondary structure , 
  with the initial 90 residues ( 15861676 ) likely to form two helices 
  ( 61 ) . sequence alignment of 
  the mlp1-nbd peptide with mlp1 peptides from other saccharomyces 
  species highlights key hydrophobic residues within the first 90 residues of 
  mlp1-nbd that have been evolutionarily conserved and perhaps retained because 
  they form -helical secondary structure or serve as functional contact 
  sites . it will be necessary to determine the structure of mlp1-nbd and its 
  complex with nab2-n to define the mlp1-nbd fold more precisely and identify 
  the residues within mlp1-nbd that make contact with the hydrophobic patch and 
  residue phe in nab2-n . analysis of the binding between nab2-n and mlp1-nbd indicates that mlp1-nbd 
  binds to nab2-n with a relatively weak affinity that is in the micromolar 
  range ( kd  1.3 m ) . apparent binding 
  affinities between the nuclear transport factor , ntf2 , or the mrna receptor 
  heterodimer , tap - p15 , and fg repeat - containing nucleoporins have been measured 
  and are also in the micromolar range 
  ( 59 , 
  60 ) . for example , ntf2 binds 
  to fg repeat nucleoporins with an apparent kd of 14 
  m and tap - p15 binds to fg repeat nucleoporins with an apparent 
  kd of 411 m 
  ( 59 , 
  60 ) . therefore , the binding 
  constant of 1.3 m observed between nab2 and mlp1 is comparable 
  with the binding constants measured between other nuclear transport factors 
  and fg - nucleoporins . like the low - affinity transport factor - nucleoporin 
  interactions , the rather weak nab2/mlp1 interaction is therefore likely to be 
  transient in vivo . in this way , nab2 may bind transiently to mlp1 
  with a relatively high off - rate consistent with rapid transport of the 
  properly assembled mrna export complex . quantitative analysis of the interaction between nab2-n - f73d and mlp1-nbd 
  shows that mlp1-nbd binding to nab2-n - f73d is too weak to measure . this result 
  supports previous data showing that ct - mlp1 can not interact with nab2-n - f73d 
  in vitro and confirms that nab2 residue phe is critical 
  for the nab2/mlp1 interaction . in addition , we find that mlp1-nbd binds to a 
  second nab2 mutant , nab2-n - f73w , with 1.5-fold higher affinity 
  ( kd  0.8 m ) compared with wild - type 
  protein ( kd  1.3 m ) . this result 
  strengthens the idea that large hydrophobic residues within nab2-n are 
  important for binding to mlp1 and underscores the hydrophobic nature of the 
  nab2/mlp1 interaction . a key finding of this study is that expression of nab2 f73d and the 
  consequent disruption of the nab2/mlp1 interaction in vivo causes 
  nuclear accumulation of poly(a ) rna and that this nab2 f73d mutant 
  displays genetic interactions with alleles of two genes essential for mrna 
  export , mex67 and yra1 . this result indicates that the 
  nab2/mlp1 interaction is functionally important and contributes to mrna 
  export . nab2 f73d cells show a 2.5-fold increase in nuclear 
  accumulation of poly(a ) rna relative to wild - type nab2 cells , but do 
  not exhibit a detectable growth defect . a potential explanation for this 
  result is that the nab2/mlp1 interaction increases the rate and/or efficiency 
  of one step of the mrna export pathway that is not normally rate - limiting and 
  therefore the nab2/mlp1 interaction is not absolutely required for cell growth 
  in a wild - type background . mex67 - 5 nab2 f73d cells , however , show a 
  1.7-fold increase in nuclear accumulation of poly(a ) rna relative to 
  mex67 - 5 nab2 cells and clearly exhibit a decrease in cell growth . one 
  possible explanation for the growth defect in mex67 - 5 nab2 f73d cells 
  is that the nab2/mlp1 interaction becomes critical for a cellular function , 
  such as efficient mrna export , when additional steps in mrna export and/or 
  mrna metabolism are compromised due to impairment of the mex67 mrna export 
  receptor . a second potential explanation for the growth defect in mex67 - 5 
  nab2 f73d cells is that a threshold number of cells that exhibit mrna 
  export defects within the population must be reached before any effects on the 
  overall growth properties of the culture are observed . thus , the 30% of 
  nab2 f73d mex67 - 5 cells that show nuclear accumulation of poly(a ) rna 
  may be sufficient to impact the overall growth properties of the cell 
  population , whereas the 8% of nab2 f73d cells that show nuclear 
  poly(a ) rna accumulation may not be sufficient . further study will be required 
  to ascertain precisely how defects in cell growth are linked to impaired mrna 
  export . the analysis of the nab2 f73d mutant presented here and previous work on 
  components of the mrna export pathway suggest a model for nab2 function in 
  mrna export in which nab2 helps to target mrna to the nuclear pore for export 
  ( fig . mrna is co - transcriptionally loaded with the mrna export 
  adaptor , yra1 ( 21 , 
  62 ) , and the poly(a ) tails of 
  the mrna are specifically bound by nab2 during or after polyadenylation 
  ( 3436 ) . yra1 binds to the mrna export protein , mex67 
  ( 18 , 
  19 ) , and recruits the 
  mex67/mtr2 heterodimeric receptor to the mrna 
  ( 16 ) . mex67/mtr2 then 
  interacts with fg - nucleoporins at the pore to translocate the mrna to the 
  cytoplasm ( 17 ) . alongside yra1 
  and mex67 , nab2 binds to mlp1 at the pore 
  ( 38 ) , via interaction between 
  the nab2-n hydrophobic patch and the mlp1-nbd , to help concentrate the mrna at 
  the pore for interaction with other mrna export factors and transport to the 
  cytoplasm . the assembled mrna complex containing nab2 is then translocated 
  through the npc channel by mex67/mtr2 , which interacts with fg - nucleoporins in 
  the channel ( 17 , 
  63 ) . upon reaching the 
  cytoplasmic face of the nuclear pore , the mrna export complex encounters the 
  dead - box helicase , dbp5 ( 64 , 
  65 ) , which is bound to and 
  activated by the nucleoporin , gle1 
  ( 6669 ) , 
  and dbp5 remodels the mrnp to trigger dissociation of nab2 from the rna 
  ( 70 ) , release of mex67 , and 
  disassembly of the complex 
  ( 71 ) . importantly , examination 
  of dbp5 function suggests that disassembly of the mrna export complex is 
  probably a rate - limiting step in mrna export 
  ( 70 , 
  71 ) . in a more detailed mrna 
  export model , several other mrna export adaptors , hnrnps , and mrna processing 
  factors can be added that aid in the concentration of mrna at the nuclear face 
  of the pore , the translocation of that mrna through the pore and its 
  remodeling and disassembly at the cytoplasmic face of the pore . figure 7.models for nab2 function in mrna export and the observed growth and mrna 
  export defects in mex67 - 5 nab2 f73d cells at the permissive 
  temperature . a , model for nab2 function in mrna export initially , mrna is 
  co - transcriptionally loaded with the mrna export adaptor , yra1 
  ( 21 , 
  62 ) , and bound by nab2 at the 
  poly(a ) tail 
  ( 3436 ) . yra1 then binds to the mrna export protein , mex67 
  ( 18 , 
  19 ) , recruiting the mex67/mtr2 
  heterodimeric receptor to the mrna 
  ( 16 ) and mex67/mtr2 interacts 
  with fg - nucleoporins at the pore to translocate the mrna to the cytoplasm 
  ( 17 ) . alongside yra1 and 
  mex67 , nab2 binds to mlp1 at the pore 
  ( 38 ) , via the nab2-n / mlp1-nbd 
  interaction . the assembled mrna complex containing nab2 is then translocated 
  through the npc channel by mex67/mtr2 
  ( 17 , 
  63 ) . upon reaching the 
  cytoplasmic face of the nuclear pore , the mrna export complex encounters the 
  dead - box helicase , dbp5 ( 64 , 
  65 ) , tethered and activated by 
  the nucleoporin , gle1 
  ( 6669 ) , 
  which remodels the mrnp to trigger dissociation of nab2 from the rna 
  ( 70 ) , release of mex67 , and 
  disassembly of the complex 
  ( 71 ) . b , model to 
  explain the observed growth and mrna export defects in mex67 - 5 nab2 
  f73d cells at the permissive temperature in which combined impairment of 
  mex67 and nab2 reduces mrna export . here , the mex67 - 5 mutant , which can not 
  interact with mtr2 and fg - nucleoporins at the non - permissive temperature 
  ( 8 , 
  16 ) , may also be impaired in 
  binding to fg - nucleoporins at the permissive temperature , potentially 
  resulting in a moderate reduction in the rate of translocation . similarly , the 
  nab2 f73d mutant , which can not interact with mlp1 , may reduce the rate of mrna 
  concentration at the pore . combination of the reduction in mrna translocation 
  rate produced by mex67 - 5 and reduction in the mrna concentration rate caused 
  by nab2 f73d could become rate - limiting and result in a decreased rate of mrna 
  export coupled with nuclear accumulation of poly(a ) rna . models for nab2 function in mrna export and the observed growth and mrna 
  export defects in mex67 - 5 nab2 f73d cells at the permissive 
  temperature . a , model for nab2 function in mrna export in which 
  nab2 helps to target mrna to the nuclear pore for export . initially , mrna is 
  co - transcriptionally loaded with the mrna export adaptor , yra1 
  ( 21 , 
  62 ) , and bound by nab2 at the 
  poly(a ) tail 
  ( 3436 ) . yra1 then binds to the mrna export protein , mex67 
  ( 18 , 
  19 ) , recruiting the mex67/mtr2 
  heterodimeric receptor to the mrna 
  ( 16 ) and mex67/mtr2 interacts 
  with fg - nucleoporins at the pore to translocate the mrna to the cytoplasm 
  ( 17 ) . alongside yra1 and 
  mex67 , nab2 binds to mlp1 at the pore 
  ( 38 ) , via the nab2-n / mlp1-nbd 
  interaction . the assembled mrna complex containing nab2 is then translocated 
  through the npc channel by mex67/mtr2 
  ( 17 , 
  63 ) . upon reaching the 
  cytoplasmic face of the nuclear pore , the mrna export complex encounters the 
  dead - box helicase , dbp5 ( 64 , 
  65 ) , tethered and activated by 
  the nucleoporin , gle1 
  ( 6669 ) , 
  which remodels the mrnp to trigger dissociation of nab2 from the rna 
  ( 70 ) , release of mex67 , and 
  disassembly of the complex 
  ( 71 ) . b , model to 
  explain the observed growth and mrna export defects in mex67 - 5 nab2 
  f73d cells at the permissive temperature in which combined impairment of 
  mex67 and nab2 reduces mrna export . here , the mex67 - 5 mutant , which can not 
  interact with mtr2 and fg - nucleoporins at the non - permissive temperature 
  ( 8 , 
  16 ) , may also be impaired in 
  binding to fg - nucleoporins at the permissive temperature , potentially 
  resulting in a moderate reduction in the rate of translocation . similarly , the 
  nab2 f73d mutant , which can not interact with mlp1 , may reduce the rate of mrna 
  concentration at the pore . combination of the reduction in mrna translocation 
  rate produced by mex67 - 5 and reduction in the mrna concentration rate caused 
  by nab2 f73d could become rate - limiting and result in a decreased rate of mrna 
  export coupled with nuclear accumulation of poly(a ) rna . 7b presents a 
  plausible model to account for the observed growth and mrna export defects in 
  mex67 - 5 nab2 f73d cells . we envisage that the mex67 - 5 mutant , which 
  can not interact with mtr2 and fg - nucleoporins at the non - permissive 
  temperature ( 8 , 
  16 ) , may also be impaired in 
  binding to fg - nucleoporins even at the permissive temperature . as the 
  interaction between mex67 and the fg - nucleoporins is necessary for the 
  translocation step of mrna export 
  ( 17 ) , the mex67 - 5 mutant may 
  reduce the rate of translocation through the nuclear pore . at the permissive 
  temperature , this modest reduction in the rate of translocation produced by 
  mex67 - 5 may not impact the overall rate of mrna export as the translocation 
  step is probably not normally rate - limiting . similarly , the nab2 f73d mutant , 
  which can not interact with mlp1 , may reduce the rate of mrna concentration at 
  the pore . at the permissive temperature , this moderate reduction in the rate 
  of mrna concentration produced by nab2 f73d may not affect the total rate of 
  mrna export as the concentration step is also probably not normally 
  rate - limiting . however , the cumulative effect of impairing both mrna 
  translocation and concentration steps could be more marked and reduce the 
  overall rate of mrna export . at the permissive temperature , in mex67 - 5 
  nab2 f73d cells , the combined reduction in the mrna translocation rate 
  produced by mex67 - 5 and the reduction in mrna concentration rate caused by 
  nab2 f73d could become rate - limiting and result in a decreased rate of mrna 
  export coupled with nuclear accumulation of poly(a ) rna . alternatively , in 
  mex67 - 5 nab2 f73d cells , malformed mrnp complexes containing nab2 
  f73d and mex67 - 5 may be generated that are not recognized and removed by the 
  surveillance machinery because nab2 f73d does not bind to mlp1 efficiently . the combined data presented here highlight the importance of the nab2/mlp1 
  interaction in vivo and underscore the extent of cooperation between 
  mrna export factors , nab2 , yra1 , and mex67 , that is necessary for efficient 
  mrna export . in addition , the data suggest that although the mrna export 
  system in yeast can tolerate single component mutations , such as nab2 
  f73d , double component mutations , like mex67 - 5 nab2 f73d , 
  produce a growth defect and significant nuclear accumulation of poly(a ) rna , 
  consistent with the step in the export pathway in which they participate 
  becoming rate - limiting , even under permissive conditions . the results of this 
  study are therefore consistent with a model in which nab2 helps to target the 
  mrnp complex to the nuclear pore via interaction with mlp1 to facilitate mrna 
  export . in this model , it is clear that nab2 acts in concert with other mrna 
  export factors , particularly yra1 and mex67 , to form an mrnp that is competent 
  of export . future studies on the genetic and physical interactions between 
  nab2 and other mrna export components will help to elucidate the underlying 
  mechanism of mrna export in eukaryotic organisms .
nuclear export of mrna requires several key mrna - binding proteins that 
  recognize and remodel the mrna and target it for export via interactions with 
  the nuclear pore complex . in saccharomyces cerevisiae , the shuttling 
  heterogeneous nuclear ribonucleoprotein , nab2 , which is essential for mrna 
  export , specifically recognizes poly(a ) rna and binds to the nuclear 
  pore - associated protein , myosin - like protein 1 ( mlp1 ) , which functions in mrna 
  export and quality control . 
 specifically , the n - terminal domain of nab2 
  ( nab2-n ; residues 197 ) interacts directly with the c - terminal globular 
  domain of mlp1 ( ct - mlp1 : residues 14901875 ) . 
 recent structural and 
  binding studies focused on nab2-n have shown that nab2-n contains a 
  hydrophobic patch centered on phe73 that is critical for 
  interaction with mlp1 . 
 engineered amino acid changes within this patch disrupt 
  the nab2/mlp1 interaction in vitro . given the importance of nab2 and 
  mlp1 to mrna export , we have examined the nab2/mlp1 interaction in greater 
  detail and analyzed the functional consequences of disrupting the interaction 
  in vivo . 
 we find that the nab2-binding domain of mlp1 ( mlp1-nbd ) maps 
  to a 183-residue region ( residues 15861768 ) within ct - mlp1 , binds 
  directly to nab2 with micromolar affinity , and confers nuclear accumulation of 
  poly(a ) rna . 
 furthermore , we show that cells expressing a nab2 f73d mutant 
  that can not interact with mlp1 exhibit nuclear accumulation of poly(a ) rna and 
  that this nab2 f73d mutant genetically interacts with alleles of two 
  essential mrna export genes , mex67 and yra1 . 
 these data 
  provide in vivo evidence for a model of mrna export in which nab2 is 
  important for targeting mrnas to the nuclear pore for export .