A role for the dynamin-like protein Vps1 during endocytosis in yeast

Abstract

SummaryDynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has raised questions over the general applicability of the current yeast model of endocytosis, and has also precluded studies using well-developed methods in yeast, to further our understanding of the mechanism of dynamin function during endocytosis. Here, we investigate the yeast dynamin-like protein Vps1 and demonstrate a transient burst of localisation to sites of endocytosis. Using live-cell imaging of endocytic reporters in strains lacking vps1, and also electron microscopy and biochemical approaches, we demonstrate a role for Vps1 in facilitating endocytic invagination. Vps1 mutants were generated, and analysis in several assays reveals a role for the C-terminal self-assembly domain in endocytosis but not in other membrane fission events with which Vps1 has previously been associated.

Ultrastructural Imaging of Endocytic Sites in Saccharomyces cerevisiae by Transmission Electron Microscopy and Immunolabeling

“…6E). Such repetitive densities have been observed in wild-type cells previously (Smaczynska-de Rooij et al, 2010). In our hands these densities were only clearly visible in the cap1 Δ knockout strain, possibly due to accumulation of endocytic invaginations in a specific stage of internalization.…”

“…1D), as we described previously (Kishimoto et al, 2011). In wild-type cells the membrane invaginates perpendicularly to the plasma membrane with less than 30° deviation (Smaczynska-de Rooij et al, 2010; Kishimoto et al, 2011). The constriction of the tubule region brings the opposing membranes within fusion range and at the same time creates the lumen of the future endocytic vesicle.…”

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). …”

Insights into dynamin-associated disorders through analysis of equivalent mutations in the yeast dynamin Vps1

“…In other studies we have observed abnormal endocytic phenotypes caused by vps1 alleles in cells that appear to carry out other Vps1 functions appropriately 253340. We therefore aimed to determine whether any of the disease mutations were able to affect the progression of individual endocytic events.…”

“…Self assembly assays: method adapted from 25. 10 µM Vps1 (or mutant variant) was added to liposome buffer at the concentrations shown and centrifuged at 313,000g for 15 min to remove any aggregated protein.…”