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. 2004 Dec;15(12):5528-37.
doi: 10.1091/mbc.e04-08-0666. Epub 2004 Sep 15.

Multivesicular body-ESCRT components function in pH response regulation in Saccharomyces cerevisiae and Candida albicans

Wenjie Xu  1 , Frank J Smith Jr, Ryan Subaran, Aaron P Mitchell
Affiliations

Multivesicular body-ESCRT components function in pH response regulation in Saccharomyces cerevisiae and Candida albicans

Wenjie Xu et al. Mol Biol Cell. 2004 Dec.

Abstract

The ESCRT-I, -II, and -III protein complexes function to create multivesicular bodies (MVBs) for sorting of proteins destined for the lysosome or vacuole. Prior studies with Saccharomyces cerevisiae have shown that the ESCRT-III protein Snf7p interacts with the MVB pathway protein Bro1p as well as its homolog Rim20p. Rim20p has no role in MVB formation, but functions in the Rim101p pH-response pathway; Rim20p interacts with transcription factor Rim101p and is required for the activation of Rim101p by C-terminal proteolytic cleavage. We report here that ESCRT-III proteins Snf7p and Vps20p as well as all ESCRT-I and -II proteins are required for Rim101p proteolytic activation in S. cerevisiae. Mutational analysis indicates that the Rim20p N-terminal region interacts with Snf7p, and an insertion in the Rim20p "Bro1 domain" abolishes this interaction, as determined with two-hybrid assays. Disruption of the MVB pathway through mutations affecting non-ESCRT proteins does not impair Rim101p processing. The relationship between the MVB pathway and Rim101p pathway is conserved in Candida albicans, because mutations in four ESCRT subunit genes abolish alkaline pH-induced filamentation, a phenotype previously seen for rim101 and rim20 mutants. The defect is suppressed by expression of C-terminally truncated Rim101-405p, as expected for mutations that block Rim101p proteolytic activation. These results indicate that the ESCRT complexes govern a specific signal transduction pathway and suggest that the MVB pathway may provide a signal that regulates pH-responsive transcription.

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Figures

Figure 1.
Figure 1.
(A) Two-hybrid assays of Snf7p-Rim20p interaction. Strain Y190, carrying pWX287 (GBD-Snf7p fusion) or pAS2-1 (GBDvector), was mated with strain Y187, carrying pWX36 (GAD-Rim20p fusion) or pACT2 (GAD-vector), two-hybrid reporter GAL1-lacZ expression was visualized on X-gal plates. (B) Processing of epitope-tagged Rim101-HA2p in wild-type and mutant strains. Extracts from yeast strains grown in YPD medium were analyzed on an anti-HA Western blot to visualize Rim101-HA2p. The masses of Rim101-HA2p forms, indicated along the right margin, were estimated by comparison with size standards. The strains included WXY169 (RIM101-HA2), WXY219 (RIM101-HA2 rim20Δ), and WXY306 (RIM101-HA2 snf7Δ).
Figure 3.
Figure 3.
Processing of epitope-tagged Rim101-HA2p in wild-type and mutant strains. Extracts from yeast strains grown in YPD medium were analyzed on an anti-HA Western blot to visualize Rim101-HA2p. The strains included the wild-type WXY169 (lane wt), and mutants derived from WXY169 by deletion of the entire ORF of each respective gene as labeled. ESCRT-I, -II, and -III complex subunits are designated -I, -II, and -III, respectively.
Figure 2.
Figure 2.
Processing of epitope-tagged Rim101-HA2p in strain WXY219 (RIM101-HA2 rim20Δ) carrying a complementing wild-type RIM20 plasmid pWX272 (lane wt), pRS314 (vector control, lane v), or various rim20 insertion mutant plasmids. The RIM20 insertion mutations include those causing premature nonsense mutations (A; mutants described in Table 1) or 5 codon in-frame insertions (B; mutants described in Table 2).
Figure 4.
Figure 4.
Filamentation of C. albicans wild-type and mutant strains. Colonies grown for 3 d at 37°C on M199 pH 8 plates were photographed. Similar results were obtained with three independently isolated insertion mutants for each gene. Hisreference strain DAY286 (A) was compared with Hismutant strains carrying insertions in RIM101 (B) and other genes as indicated (C-J). His+ derivatives of each mutant were created with integrated HIS1:RIM101 plasmids (K-N) or integrated HIS1:RIM101-405 plasmids (P-S). The RIM101-405 allele expresses a truncated Rim101p derivative that permits alkaline pH-induced filamentation in the absence of upstream processing pathway components (Davis et al., 2000).
Figure 5.
Figure 5.
Relationship between the Rim101p and MVB pathways. The ESCRT-I, ESCRT-II, and Snf7p-Vps20p complexes are required for both MVB formation and Rim101p activation. Homologous proteins Bro1p and Rim20p interact with Snf7p and function in the MVB and Rim101p pathways, respectively. The MVB pathway delivers cargo proteins to the vacuole that include membrane proteins destined for degradation and proteases. Rim101p represses diverse genes including PRB1, which specifies a major vacuolar endopeptidase. Changes in overall ESCRT activity may modulate Rim101p and MVB pathway activity in parallel. In addition, the two pathways may compete for limiting ESCRT activity.
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