Distinct segregation patterns of yeast cell-peripheral proteins uncovered by a method for protein segregatome analysis

Abstract

Protein segregation contributes to various cellular processes such as polarization, differentiation, and aging. However, the difficulty in global determination of protein segregation hampers our understanding of its mechanisms and physiological roles. Here, by developing a quantitative proteomics technique, we globally monitored segregation of preexisting and newly synthesized proteins during cell division of budding yeast, and identified crucial domains that determine the segregation of cell-peripheral proteins. Remarkably, the proteomic and subsequent microscopic analyses demonstrated that the flow through the bud neck of the proteins that harbor both endoplasmic reticulum (ER) membrane-spanning and plasma membrane (PM)-binding domains is not restricted by the previously suggested ER membrane or PM diffusion barriers but by septin-mediated partitioning of the PM-associated ER (pmaER). Furthermore, the proteomic analysis revealed that although the PM-spanning t-SNARE Sso2 was retained in mother cells, its paralog Sso1 unexpectedly showed symmetric localization. We found that the transport of Sso1 to buds was required for enhancement of polarized cell growth and resistance to cell-wall stress. Taken together, these data resolve long-standing questions about septin-mediated compartmentalization of the cell periphery, and provide new mechanistic insights into the segregation of cell-periphery proteins and their cellular functions.

Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells

“…The limitation of our segmentation method is that budding cells are accepted as two separate cells with the "whole-cell" plasma membrane without accounting for the motherdaughter cell connection through the bud neck. Therefore, the mobile pool might represent Fps1 located closer to the bud, where proteins can move between the mother and the daughter cells, which would be consistent with previous reports on multiple plasma membrane proteins are asymmetrically segregated 51 . However, further studies with bud neck labelling should be performed to verify that.…”

Two- and Three-Dimensional Tracking of MFA2 mRNA Molecules in Mating Yeast

“…These granules have dynamic structures that change in size and location during shmoo growth and are pulled to the bud/shmoo tip of the cell. [ 63 , 64 ]. MFA2 RNP velocity can be dependent on the growth of tubular ER to the shmoo.…”

How Diffusion Impacts Cortical Protein Distribution in Yeasts

“…Early hypotheses to explain such asymmetries involved a diffusion barrier at the mother-bud neck linked to cytoskeletal septin filaments found at that location [8,9]. However, subsequent work suggested instead that no barrier is needed because diffusion in the yeast plasma membrane is remarkably slow [10].…”

“…Given the very restricted lateral mobility of plasma membrane proteins in yeast, one way to obtain mother-enriched protein localization would be to restrict protein synthesis to a specific phase of the cell cycle, as discussed for cell wall proteins ( Figure 1B). Indeed, the timing of synthesis combined with slow diffusion appears to account for much of the observed asymmetry for these long-lived proteins [10].…”

“…Asymmetric accumulation of pheromone receptors has the potential to mislead mating cells about the location of a mating partner, requiring corrective mechanisms to provide accurate detection [12]. It would seem quite simple to avoid these problems, and there are instances where endocytic recycling has been tuned so as to counteract the asymmetry derived from timed synthesis in the cell cycle [10]. Why, then, are apparently unproductive asymmetries maintained?…”