Figure 3.—

Inactivation of ubl-5 compromises signaling in the UPR^mt. (A) Fluorescent photomicrographs of wild-type (N2) and zc32 mutant animals transgenic for the UPR^mt reporters hsp-6∷gfp and hsp-60∷gfp or for the UPR^er reporter hsp-4∷gfp. Animals developed from founders placed on mock RNAi or ubl-5(RNAi) plates at 20° and were shifted, where indicated, to the nonpermissive temperature (25°) for 24 hr before analysis. The animals in the left panels were exposed to genomic-based RNAi whereas those on the right were exposed to cDNA-based RNAi constructs derived from C. elegans mRNA (Ce) or C. briggsae mRNA (Cb). Where indicated, the hsp-4∷gfp reporter was activated by exposure to the ER-stress-inducing drug tunicamycin [note that ubl-5(RNAi) does not affect the induction of hsp-4∷gfp]. (B) Photomicrographs of progeny of wild-type (N2) or zc32 founder animals that developed on mock or ubl-5(RNAi) plates maintained for 16 hr at the permissive temperature of 20° (to allow egg laying by the founder) and shifted to the nonpermissive temperature of 25° for 3 days. The number of F₁ progeny that reached developmental stage ≥L4 (mean ±SEM)/F₀ hermaphrodite is indicated below the images. (C) Immunoblot of GFP reporter in hsp-60∷gfp transgenic wild-type, zc32 mutant, or animals exposed to the indicated mitochondrial stress-inducing RNAi in the presence or absence of ubl-5(RNAi). The anti-HDEL blot, which detects C. elegans BiP, serves as a loading control. (D) Northern blot of endogenous hsp-60 and hsp-6 RNA in animals that developed on mock RNAi, spg-7(RNAi) (to induce mitochondrial stress), and a combination of spg-7(RNAi) and ubl-5(RNAi). Samples were processed 72 and 96 hr after seeding the RNAi plates with eggs. Ribosomal RNAs stained with ethidium bromide serve as a loading control. (E) Immunoblot of GFP, as in C. The GFP protein encoded by the hsp-60∷gfp(zcIs9) transgenic UPR^mt reporter (hsp-60∷GFP) and the UBL-5Cb∷GFP fusion protein encoded by the rescuing transgenic allele of ubl-5^Cb∷gfp(zcIs22) are indicated. The asterisk marks an immunoreactive protein fragment found in ubl-5^Cb∷gfp(zcIs22) animals, which is likely an in vitro proteolytic fragment of the fusion protein. The anti-HDEL blot, which detects C. elegans BiP, serves as a loading control.