A Dynamin-Actin Interaction Is Required for Vesicle Scission during Endocytosis in Yeast

Abstract

SummaryActin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that—like wild-type Vps1—it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

A Charge Swap mutation E461K in the yeast dynamin Vps1 reduces endocytic invagination

“…We generated equivalent charge swap mutations in yeast Vps1 to determine whether an interaction with actin was conserved and if so, whether it was important for any or all functions of the protein. 10 Vps1 was shown to bind actin and the vps1 RR-EE mutation reduced actin binding as predicted. A detailed analysis of the scission defects caused by this mutation are described in our recent paper.…”

“…A reduced level of Rvs167 suggests a defect early in invagination rather than at the later scission stage when, as observed in the wild type situation or in the vps1 RR-EE mutant, higher levels of Rvs167 are observed. 10 Taken together the data indicate that a form of vps1 carrying a mutation E461K in the proposed actin binding helix of the central stalk domain is still able to bind actin. However, actin binding does not lead to increased oligomerization of Vps1 as observed with wild-type protein.…”

“…A detailed analysis of the scission defects caused by this mutation are described in our recent paper. 10 One of the other mutations, vps1 E461K (Fig. 1A), showed similar actin binding to wild type Vps1 in a high-speed pelleting assay (Fig.…”

“…10 However analysis of fluid phase uptake indicated a delay in endocytosis. Using electron microscopy of cells fixed by high pressure freezing, a high proportion of short/ shallow invaginations were observed with a mean length of 18 § 7nm n D 50 invaginations, compared to 50-60 nm for invagination length in wild type cells.…”

Clathrin-mediated endocytosis in budding yeast at a glance

“…The lack of Rvs161-Rvs167 and Bzz1 results in an improper geometry of invagination (Kishimoto et al, 2011). Similarly, deletion of VPS1, whose gene product is a dynamin-like protein, results in 'yo-yo' phenotypes and misshaped invaginations (Smaczynska-de Rooij et al, 2010), consistent with a role in regulating membrane shape and scission, potentially through an F-actin-bundling activity (Palmer et al, 2015). Unlike the conventional dynamins involved in mammalian CME, and similar to other dynamin-like proteins, Vps1 does not contain a pleckstrinhomology (PH) domain.…”