Slt2 and Rim101 Contribute Independently to the Correct Assembly of the Chitin Ring at the Budding Yeast Neck in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the simultaneous absence of Slt2 and Rim101 prevents growth in nonosmotically stabilized media (F. Castrejon et al., Eukaryot. Cell 5:507-517, 2006). The double mutant slt2⌬ rim101⌬ displays altered chitin rings, together with a significant reduction in the overall levels of chitin. Cultures of this mutant lyse upon transfer to nonosmotically stabilized media, mostly through the bud, and such lysis is partially prevented by deletion of the chitinase gene (CTS1). Growth of the slt2⌬ rim101⌬ double mutant was restored by the overexpression of the GFA1 or CCT7 genes, which code for two biologically unrelated proteins. Further characterization of the mutant and its suppressors indicated that both Slt2 and Rim101 were independently required for the correct assembly of the septum machinery and that their concomitant absence reduced Chs3 accumulation at the neck, leading to lower levels of chitin. GFA1 overexpression, as well as the addition of glucosamine to the growth medium, specifically suppressed the growth defects by activating chitin synthesis at the neck and restoring the normal assembly of the chitin ring. In contrast, overexpression of CCT7, a Cct chaperonin subunit, alleviated the defect in the septum machinery without affecting chitin synthesis. Both suppressors thus act by reducing neck fragility through different mechanisms and allow growth in nonstabilized media. This work reports new roles for Slt2 and Rim101 in septum formation in budding yeast and confirms the homeostatic role of the chitin ring in the maintenance of neck integrity during cell division.

Upregulation of the PRB1 gene in the Saccharomyces cerevisiae rim101Δ mutant produces proteolytic artefacts that differentially affect some proteins

“…However, analysis of the transcriptional response mediated by calcofluor in this mutant (Figure 1; and J. Perez, unpublished observations) indicates that signalling is reduced in the absence of a functional RIM101 pathway. This result is consistent with the mild cell wall‐associated phenotypes observed for the rim mutants (Castrejon et al , 2006; Gomez et al , 2009). The promiscuity of subtilisin‐like proteases makes it difficult to pinpoint the reasons for the different in vitro stability of two biologically related proteins, such as Chs3p and Chs4p.…”

Equivalent Mutations in the Eight Subunits of the Chaperonin CCT Produce Dramatically Different Cellular and Gene Expression Phenotypes

“…For example, as mentioned above, overexpression of CCT6 suppresses the lethality of a TOR2 mutation in yeast. 27,28 It is therefore intriguing that overexpression of CCT7 was recently reported to alleviate a defect in the septum machinery in the slt2Δ rim101Δ mutant, 35 given that only MA7 cells were found here to have elongated buds (Fig. 3).…”

Neck compartmentalization as the molecular basis for the different endocytic behaviour of Chs3 during budding or hyperpolarized growth in yeast cells

“…Calcofluor vital staining was observed in cells grown in the presence of 50 µg ml −1 Calcofluor for 2 h at 28°C (Gomez et al ., 2009). Transformant cells expressing GFP, and the mCherry or mRFP fusions were grown in SD medium supplemented with 0.2% adenine and visualized directly by fluorescence microscopy in living cells.…”

“…Most chitin is assembled in a chitin ring at the mother side of the neck. This ring, although fully dispensable for cytokinesis, becomes essential when this process is minimally altered under a variety of conditions (Gomez et al ., 2009; Roncero and Sanchez, 2010). Its synthesis depends on the activation of CSIII at the PM but also on the anchoring of this activity to the neck, both processes being directly linked to the function of Chs4 (Trilla et al ., 1997; Sanz et al ., 2004; Reyes et al ., 2007).…”