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

Abstract

SummaryYeast cells normally grow by budding, but under certain specific conditions they are also able to grow in hyperpolarized forms reminiscent of hyphal growth. During vegetative growth, the synthesis of the septum that physically separates yeast cells during cytokinesis depends on the correct assembly of the septin ring. Septins and actin patches are assembled at the neck, forming two concentric rings where the actin patch ring occupies the external-most part. This specific positioning defines a plasma membrane region at the neck from which other lateral membrane compartments are excluded. In this scenario, correct assembly of the chitin ring is dependent on the anchoring of Chs3 to the septin ring through Chs4. The anchoring of Chs3 to septins through Chs4 prevents the arrival of this protein at endocytic sites, thus reducing the endocytosis of Chs3. This allows an equilibrium to be set up between the antero-and retrograde transport of Chs3, facilitating the synthesis of the chitin ring at the neck. In contrast, hyperpolarized growth is characterized by a reduced endocytic turnover of Chs3, which in turn lead to the accumulation of Chs3 at the plasma membrane and a concomitant increase in chitin synthesis.

In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae

“…Cells expressing a Chs3 VC version lacking only the N-terminus (Chs3 210–1165 VC ) showed BiFC signals only on the membranes of intracellular vesicles and this was observed in only about 10% of all evaluated cells. The results presented so far suggest that the interaction site may be located in the region of the N-terminus between amino acid positions 1 and 209, as previously proposed based on Y2H analyses and crosslinking experiments [38]. …”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…Chs3 is necessary because no extra chitin is made in pheromone-treated chs3D cells, and the response is either abolished or much smaller in chs5D, chs6D, and chs4D cells, indicating that the machinery for trafficking and activation of Chs3 is required (Orlean 1987;Roncero et al 1988;Bulawa 1993;Santos and Snyder 1997;Bulik et al 2003). Consistent with its role in chitin deposition, Chs3 is localized at the periphery of the mating projection, and it remains there because it is not subject to endocytic turnover as it is in budding cells (Santos and Snyder 1997;Sacristan et al 2012). Although the extra chitin synthesis in response to a-factor is presumably driven by the increased amount of UDP-GlcNAc made during the pheromone response (Orlean et al 1985;Bulik et al 2003), the mechanism behind pheromone-stimulated chitin synthesis by Chs3 is unclear.…”

Maintaining protein homeostasis: early and late endosomal dual recycling for the maintenance of intracellular pools of the plasma membrane protein Chs3

“…Chs3 acts at the PM, and therefore we checked whether Chs3 ubiquitination occurred here by testing the status of Chs3 ubiquitination in different mutants that showed higher levels of Chs3 at the PM because of the reduced levels of Chs3 endocytosis (Reyes et al. , 2007; Sacristan et al. , 2012).…”