Bem3, a Cdc42 GTPase-Activating Protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

Abstract

SummaryCell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking.

RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking

“…2A and B). 37,38 Our results add further layers of complexity to the regulation of the exocyst complex, as well as highlight the commonalities observed between yeast and mammals in relation to secretory vesicle traffic regulation.…”

“…When the period of apical growth was experimentally extended, the size of the Bem3 compartment increased substantially, suggesting that the Bem3-labeled compartment plays a role in apical bud growth. 37 A similar compartment called the Spitzenkorper has been observed during hyphal bud elongation in filamentous fungi-a recycling station that provides a ready pool of proteins and lipids for rapid membrane expansion during hypha development. 43 Figure 1.…”

“…Moreover, in a recycling deficient strain (rcy1D), the cumulative size of this internal Bem3 compartment is significantly higher compared with wild-type strains. 37 Both these results suggest that the Bem3-labeled endomembrane is a recycling compartment.…”

“…40,42 A recent study from our lab established that Bem3 also localizes to an intracellular compartment that is distinctly visible during early stages of the cell cycle. 37 Indeed, electron micrographs revealed that Bem3 occupies a cluster of vesicles polarized toward the emerging bud ( Fig. 2A).…”

“…On the other hand, overexpression of Bem3 led to a massive accumulation of internal membranous structures that recruited and retained Sec4, the Rab GTPase regulating secretory vesicle traffic in budding yeast. 37 At this point it is important to note that Bem3 overexpression for extended periods of time is lethal, the terminal phenotype being the failure to establish polarity and generate a bud. 44 We postulate that in addition to Sec4, Bem3 overexpression leads to the accumulation of other key secretory players in internal structures.…”