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. 2004 Dec;3(6):1609-18.
doi: 10.1128/EC.3.6.1609-1618.2004.

Snf7p, a component of the ESCRT-III protein complex, is an upstream member of the RIM101 pathway in Candida albicans

Amy L Kullas  1 , Mingchun Li, Dana A Davis
Affiliations

Snf7p, a component of the ESCRT-III protein complex, is an upstream member of the RIM101 pathway in Candida albicans

Amy L Kullas et al. Eukaryot Cell. 2004 Dec.

Abstract

The success of Candida albicans as an opportunistic pathogen is based in part on its ability to adapt to diverse environments. The RIM101 pathway governs adaptation to neutral-alkaline environments and is required for virulence. Analysis of a genomic two-hybrid study conducted with Saccharomyces cerevisiae revealed that components involved in multivesicular bodies (MVB) transport may interact with RIM101 pathway members. Thus, we hypothesized that these proteins may function in the RIM101 pathway in C. albicans. We identified C. albicans homologs to S. cerevisiae Snf7p, Vps4p, and Bro1p and generated mutants in the cognate gene. We found that snf7Delta/Delta mutants, but not vps4Delta/Delta nor bro1Delta/Delta mutants, had phenotypes similar to, but more severe than, those of RIM101 pathway mutants. We found that the constitutively active RIM101-405 allele partially rescued snf7Delta/Delta mutant phenotypes. The vps4Delta/Delta mutant had subtle phenotypes, but these were not rescued by the RIM101-405 allele. Further, we found that the snf7Delta/Delta, vps4Delta/Delta, and bro1Delta/Delta mutants did not efficiently localize the vital dye FM4-64 to the vacuole and that it was often accumulated in an MVB-like compartment. This phenotype was not rescued by RIM101-405 or observed in RIM101 pathway mutants. These results suggest that Snf7p may serve two functions in the cell: one as a RIM101 pathway member and one for MVB transport to the vacuole.

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Figures

FIG. 1.
FIG. 1.
A genomic two-hybrid screening identified proteins that may interact with members of the RIM101 pathway in S. cerevisiae (18). These studies suggested that Snf7p and Vps4p interact with each other and with Rim20p. Further, they suggest that Snf7p also interacts with Rim13p. Vps4p can be immunoprecipitated with Snf7p and the Rim20p homolog Bro1p (16). Gray lines represent a two-hybrid interaction; gray double-sided arrows represent an interaction identified by tandem affinity purification (16). Numbers in parentheses represent the total number of two-hybrid interactions observed for a given protein.
FIG. 2.
FIG. 2.
Confirmation of mutants. PCR genotyping for the snf7Δ/Δ (A), vps4Δ/Δ (B), and bro1Δ/Δ (C) mutants was performed using primers that flank the gene of interest; the results of representative PCR analysis of genomic DNA from the wild type (lanes 1; BWP17 in each case), a heterozygous mutant (lanes 2; DAY530, DAY531, and DAY648, respectively), and a homozygous mutant (lanes 3; DAY534, DAY537, and DAY653, respectively) are shown. The wild-type (*), ARG4 allele (a), and URA3 allele (u) bands are noted to the left of each sample.
FIG. 3.
FIG. 3.
Growth phenotypes of ESCRT-III mutants. (A) The locations of the strains analyzed were as follows: wild-type (DAY286), vps4Δ/Δ (DAY537), snf7Δ/Δ (DAY534), bro1Δ/Δ (DAY653), rim20Δ/Δ (DAY23), and rim101Δ/Δ (DAY5). (B to D) Strains were grown on YPD (B), YPD (pH 9) (C), and YPD plus LiCl (D) for 2 days at 37°C prior to photographing.
FIG. 4.
FIG. 4.
Growth phenotypes by spot dilutions. (A) His− wild-type (DAY286), rim101Δ/Δ (DAY5), snf7Δ/Δ (DAY534), and rim20Δ/Δ (DAY23) strains were serially diluted fivefold and plated on YPD, YPD at pH 9, and YPD plus LiCl and grown at 37°C. (B) Prototrophic wild-type (DAY185), rim101Δ/Δ (DAY25), snf7Δ/Δ SNF7 (DAY761), snf7Δ/Δ (DAY763), snf7Δ/Δ RIM101-405 (DAY546), and snf7Δ/Δ RIM101 (DAY544) strains were serially diluted fivefold and plated on YPD, YPD at pH 9, and YPD plus LiCl and grown at 37°C. Plates were grown for 24 h prior to photographing.
FIG. 5.
FIG. 5.
Alkaline-induced filamentation of ESCRT-III mutants. (A) Wild-type (DAY286), rim101Δ/Δ (DAY5), snf7Δ/Δ (DAY534), vps4Δ/Δ (DAY537), bro1Δ/Δ (DAY653), and rim20Δ/Δ (DAY23) strains were grown overnight in YPD and spotted onto M199 medium (pH 8). (B) Complementation studies of the snf7Δ/Δ mutant. Wild-type (DAY185), rim101Δ/Δ (DAY25), snf7Δ/Δ RIM101 (DAY544), snf7Δ/Δ RIM101-405 (DAY546), snf7Δ/Δ SNF7 (DAY761), and snf7Δ/Δ (DAY763) strains were grown overnight in YPD and spotted onto M199 medium (pH 8). Plates were incubated at 37°C for 5 days prior to photographing.
FIG. 6.
FIG. 6.
Western blot analysis for Rim101p processing. Wild-type (DAY492), rim13Δ/Δ (DAY643), and snf7Δ/Δ (DAY568) strains that express Rim101-V5p were grown 4 h at pH 4 and 7. Protein was purified and separated by SDS-8% PAGE. Gels were transferred to nitrocellulose and probed with anti-V5 horseradish peroxidase (Invitrogen).
FIG. 7.
FIG. 7.
FM4-64 staining in ESCRT-III mutants. Wild-type (DAY286), snf7Δ/Δ (DAY534), vps4Δ/Δ (DAY537), and bro1Δ/Δ (DAY653) strains were incubated 30 min on ice in M199 medium (pH 8) containing 16 mM FM4-64 (time = 0). Cells were then pelleted, washed, and resuspended in warm M199 medium (pH 8) lacking FM4-64 (time = postincubation). Cells were incubated at 37°C, and at 45-, 90-, and 150-min intervals aliquots were taken and transferred to ice-cold tubes containing sodium azide and sodium fluoride. Photographs are representative of three independent samples. pi, postincubation.
FIG. 8.
FIG. 8.
FM4-64 staining complemented snf7Δ/Δ strains and RIM101 pathway mutants. (A) Rescue of snf7Δ/Δ mutants for FM4-64 staining: snf7Δ/Δ SNF7 (DAY761), snf7Δ/Δ (DAY763), snf7Δ/Δ RIM101 (DAY544), and SNF7Δ/Δ RIM101-405 (DAY546) strains were stained with FM4-64 as described for Fig. 7. Pictures represent samples taken after 60 min. (B) RIM101 pathway mutants and FM4-64 staining: wild-type (DAY286), rim101Δ/Δ (DAY5), rim20Δ/Δ (DAY23), rim8Δ/Δ (DAY61), rim13Δ/Δ (DAY349), and snf7Δ/Δ (DAY534) strains were stained with FM4-64 as described for Fig. 7. Pictures represent samples taken after 120 min.
FIG. 9.
FIG. 9.
Model of Snf7p function. Snf7p has two distinct functions in C. albicans. One function is to transport MVB to the vacuole during endocytosis. The second function is as a member of the RIM101 pathway, which is activated in response to neutral-alkaline environments.
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