Figure 1. Impaired UPRmt in
haf-1 Mutant Animals (A) Representative fluorescent photomicrographs of
hsp-60pr::gfp transgenic worms (reporting on the UPRmt) with a temperature-sensitive mutation (
zc32) that activates the UPRmt in an otherwise WT or
haf-1(
ok705)-deleted background. Where indicated, WT or
haf-1(
ok705) worms were raised on
spg-7(RNAi) for 3 days to induce mitochondrial misfolded protein stress. (B) A corresponding immunoblot of GFP expressed by
hsp-60pr::gfp transgenic worms in whom mitochondrial unfolded protein stress was induced (as in A). The endogenous 55 kDa ER protein, detected with an anti-HDEL monoclonal antibody (lower panel), serves as a loading control. (C) Quantitative analysis (by quantitative RT-PCR) of endogenous
hsp-60 mRNA in WT or two different
haf-1-deleted strains (
ok705 and
tm843) subjected to mitochondrial unfolded protein stress by
hsp-6(RNAi) or
spg-7(RNAi). Displayed is the mean ±SEM, n = 3, *p < 0.05. (D) Immunoblot of proteins reactive with an anti-HDEL monoclonal antibody in extracts of WT and
haf-1 mutant worms in which ER stress was induced by exposure to elevated temperature (30C) for the indicated time. The upper band, BiP (HSP-4), is a UPRER target gene, whereas the lower invariant 55 kDa band (*) serves as a loading control. (E) Fluorescent photomicrographs of CHO cells expressing GFP (upper panels) or GFP fused to aa 1-75 of HAF-1 (HAF-11-75::GFP, lower panels). The cells were costained with the vital dye MitoTracker, which stains mitochondria (middle panels). (F) Immunoblot of extracts from HEK293T cells expressing GFP (as a cytosolic marker) and C-terminally tagged HAF-1::FLAG following cellular fractionation into total lysate (T), postmitochondrial supernatant (S), and mitochondrial pellet (M). Lanes 4-8 are from mitochondria treated with hypotonic buffer to generate mitoplasts and further treated with digitonin and Proteinase K where indicated. Lanes 7 and 8 are mitoplasts incubated in Na2CO3 followed by centrifugation at 150,000 X g and separated into the pellet (P) and supernatant (Su).