Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

Abstract

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.

Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

“…HHpred (https://toolkit. tuebingen.mpg.de/hhpred) alignments of CHMP7 matched its NT to VPS25 [6], and, by aligning predicted secondary structural elements in CHMP7 to those present in the crystal structure of VPS25, we noted an evolutionarily conserved extension of the loop between the b2-b3 hairpin in the first WH domain of CHMP7 NT ( Figure S3A). Deletions through CHMP7 NT were poorly expressed ( Figures S2C and S2D), so we performed scanning mutagenesis through CHMP7 NT to identify ER localization determinants ( Figures S3B and S3C).…”

“…In C. elegans, ESCRT-II has been reported to localize to the sarcoplasmic reticulum, suggesting that the tandem WH fold may play a broader role in ER targeting [20]. We identify specific residues in the first WH domain of CHMP7 NT domain that are necessary for membrane binding, ER localization, subsequent enrichment of CHMP7 at the reforming NE, and, given CHMP7's ability to bind CHMP4 proteins [6,11], that are essential for the assembly of downstream ESCRT-III components and for ESCRT-IIIdependent NE regeneration. In the absence of membranebound CHMP7, ESCRT-III cannot assemble at the NE.…”

“…cerevisiae Chm7 was recently shown to localize to the ER [6], suggesting that this localization is evolutionarily conserved. During NE reformation, all other ESCRT-III subunits are recruited from the cytoplasm [2,3]; given that the NE is formed from the ER [9,10], a pre-existing ER localization for CHMP7 suggested a platform from which this recruitment could occur.…”

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[6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleocytoplasmic compartmentalization.

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“…CHMP7 is responsible for recruiting downstream ESCRT-III components to the reforming NE through CHMP4B. Fusion of siRNA-resistant CHMP7 NT to CHMP4B directed cytoplasmic CHMP4B to the mitotic ER and restored its enrichment at sites of annular fusion at the forming NE, in the absence of endogenous CHMP7 (Figures S2E-S2G).Analysis of the secondary structure of CHMP7 NT has revealed the presence of tandem winged helix (WH) domains [6,12], resembling those found in ESCRT-II subunits ( Figure S3A). During endosomal sorting, membrane-anchored ESCRT-II serves to recruit ESCRT-III to endosomes through interaction of the second WH domain of VPS25 with the ESCRT-III component VPS20/CHMP6 [13,14].…”