Figure 1

A-D SQST‐1::GFP is very weakly expressed and diffusely localized in the cytoplasm in wild‐type larval intestine, but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae. (A) and (C) represent DIC images of the animals in (B) and (D), respectively.

E Compared to wild‐type, SQST‐1::GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay. SQST‐1::GFP levels in bp399 mutants are greatly reduced after 12 h of starvation. 200 young adult animals for each genotype were collected for analysis.

H, I SQST‐1::GFP aggregates in rpl‐43(bp399) mutants are separable from LAAT‐1::Cherry‐labeled lysosomes under normal conditions, but are delivered to lysosomes upon starvation. Some aggregates are enclosed by lysosomes (inserts). Scale bar: 20 μm.

J-Q RNAi inactivation of let‐363, rpn‐2, cogc‐1, or hgrs‐1 suppresses the accumulation of SQST‐1::GFP aggregates in rpl‐43(bp399) mutants. (J), (L), (N), and (P): DIC images of the animals in (K), (M), (O), and (Q). Scale bar: 20 μm.

R SQST‐1::GFP levels in rpl‐43(bp399) mutants are greatly reduced by inactivation of rpt‐3 and rpn‐2. 200 young adult animals for each genotype were collected for analysis.

Figure 2

A, B SQST‐1::GFP aggregates are absent in the rpl‐43(bp399); sma‐3(wk20) mutant intestine.

C Percentage of the indicated animals with different levels of SQST‐1::GFP aggregates. S: many SQST‐1::GFP aggregates in all intestinal cells, as in rpl‐43(bp399) mutants; M: fewer SQST‐1::GFP aggregates in some but not all intestinal cells; N: no or very few SQST‐1::GFP aggregates in intestinal cells. For each genotype, > 30 animals were examined.

D SQST‐1::GFP levels in rpl‐43(bp399) mutants are dramatically decreased in a Western blot when sma‐3, lin‐35, or daf‐2 is simultaneously depleted. Loss of function of daf‐16 partially restores SQST‐1 levels in rpl‐43(bp399); daf‐2(RNAi) mutants.

E Endogenous LGG‐1 is elevated in a Western blot in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants.

F mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.

G, H No SQST‐1::GFP aggregates are observed in the lin‐35(n745); rpl‐43(bp399) mutant intestine.

I, J Numerous SQST‐1::GFP aggregates are formed in rpl‐43(bp399); lin‐15A(n767) mutants.

K Endogenous LGG‐1 levels are increased in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants.

L mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.

M, N rpl‐43(bp399); daf‐2(RNAi) mutant larvae contain no intestinal SQST‐1::GFP aggregates.

O, P

SQST‐1

::GFP aggregates are partially restored in daf‐16(mu86); rpl‐43(bp399); daf‐2(RNAi) animals.

Figure 3

A, B hsp‐4::GFP is very weakly expressed in wild‐type animals.

C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype.

D, E

Phsp‐4

::GFP expression is much higher in rpt‐3(RNAi) animals.

F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation.

H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals.

J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals.

M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.

Figure 4

Phsp‐60

::GFP is weakly expressed in wild‐type animals.

B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP.

C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.

D Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis.

E, F Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F).

G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H).

I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.