Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

Abstract

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.

Septin Organization and Functions in Budding Yeast

“…In alpha facor arrested cells, Vps1 interacts with the septins (Renz et al, 2016). Vps1 is a dynamin related protein that functions at several stages of membrane trafficking including endocytic scission (Smaczynska-de Rooij et al, 2010, 2015). …”

Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis

“…The yeast BAR domain amphiphysin protein Rvs167, interacts with Vps1 and both proteins contribute to scission of endocytic vesicles 34 . In a previous study we also demonstrated interactions between Rvs167 and Vps1 that are affected by phosphorylation of Vps1 within the Insert B region, at serine 599 close to the lysine stretch [35]. Mutation of the Vps1-Rvs167 interaction site correlated with a shorter lifetime of Rvs167 association at endocytic sites.…”

Yeast dynamin interaction with ESCRT proteins at the endosome

“…Another rationale behind the inclusion of Vps1 at the "hot spot" is that Vps1 localizes to the late endosome and interacts with PI3P in vitro (Hayden et al, 2013;Smaczynska-de et al, 2015). ESCRT-II localization to the endosome is mediated by the N-terminal region of its Vps22/Vps36 lobe.…”

“…with the membrane-remodeling protein (Figure 2), Vps1, at the endosome (Figure 3), and that Vps1 shares a redundant role with these complexes (Figure 4), namely efficient sorting of the cargo Cps1 into ILVs. At present, the biochemical targeting mechanism of Vps1 to the endosome has not been revealed, but a recent lipid binding preference test exhibited that Vps1 strongly interacts with PI3P, which is highly enriched in the endosome (Smaczynska-de et al, 2015), pointing to a lipid-binding ability of Vps1 that may facilitate its recruitment to the endosome. In light of the finding that multiple components of the ESCRT system, including Vps22, Vps36, and Vps24, bind to Vps1, we investigated the significance of these protein factors for the recruitment of Vps1 onto the endosomal membrane.…”

“…Vps1 was added to the diagram, near the periphery of the tip of the lobe for it interacts with the N-terminal GLUE and ND domains of Vps36 and Vps22, respectively. Another rationale behind the inclusion of Vps1 at the "hot spot" is that Vps1 localizes to the late endosome and interacts with PI3P in vitro (Hayden et al, 2013;Smaczynska-de et al, 2015).…”