Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis

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 dynamin-like protein Vps1 has roles at multiple stages of membrane trafficking including Golgi to vacuole transport, endosomal recycling, endocytosis and in peroxisomal fission. While the majority of the Vps1 amino acid sequence shows a high level of identity with the classical mammalian dynamins, it does not contain a pleckstrin homology domain (PH domain). The Dyn1 PH domain has been shown to bind to lipids with a preference for PI(4,5)P2 and it is considered central to the function of Dyn1 in endocytosis. The lack of a PH domain in Vps1 has raised questions as to whether the protein can function directly in membrane fusion or fission events. Here we demonstrate that the region Insert B, located in a position equivalent to the dynamin PH domain, is able to bind directly to lipids and that mutation of three lysine residues reduces its capacity to interact with lipids, and in particular with PI(4,5)P2. The Vps1 KKK-AAA mutant shows more diffuse staining but does still show some localization to compartments adjacent to vacuoles and to endocytic sites suggesting that other factors are also involved in its recruitment. This mutant selectively blocks endocytosis, but is functional in other processes tested. While mutant Vps1 can localise to endocytic sites, the mutation results in a significant increase in the lifetime of the endocytic reporter Sla2 and a high proportion of defective scission events. Together our data indicate that the lipid binding capacity of the Insert B region of Vps1 contributes to the ability of the protein to associate with membranes and that its capacity to interact with PI(4,5)P2 is important in facilitating endocytic scission

Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis

Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline–arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type ISH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast

Determination of selected drugs using tandem mass-spectrometry

<p><b>N-terminal region of Las17 mediates localization to membranes</b> (A) Constructs expressing full length Las17, PP-WCA or 2xWH1 fused to GFP were expressed in yeast and extracts tested for expression using antibodies against GFP. (B) Localization of the GFP constructs were assessed in cells co-expressing the endocytic site marker Sla2-mRFP. Top panels: full length Las17-GFP, middle panels PP-WCA-GFP; lower panels, 2xWH1-GFP. Bar = 2 μm. (C) Full length Las17-myc purified from yeast and the recombinant PP-WCA Las17 fragment (300–633) purified from <i>E</i>.<i>coli</i> were incubated with liposomes before centrifuging to assess binding. S-supernatant and P–pellet fractions. Size of MW standard markers are marked in kilodaltons.</p

Lipid binding activity of Ysc84-Nt.

<p>(A) Dot-blot assays were performed on PIP strips to determine the specificity of Ysc84 wild-type and mutants against 15 phospholipids. The membrane was incubated with 10 μM of His-tagged purified protein and proteins detected using anti-His tag antibodies. (B) Liposome co-sedimentation assay was performed using Ysc84-Nt and 70% PE, 30% PC-based liposomes supplemented in right lanes with 10% of PI(4,5)P₂. Proteins in the supernatant (S) and pellet (P) were visualized by Coomassie staining. Densitometry was used to determine the proportion of protein pelleting with liposomes (lower panel). Results are mean (±SD) of two independent experiments. Kinetics of F-actin barbed end elongation in the absence and presence of Ysc84 and PI(4,5)P₂. (C,D) In a pyrene-based fluorimetry assay pre-formed actin seeds (1 μM) were mixed with 1 μM G-actin and incubated with wild type and KK (C) or wild type and LK (D) Ysc84-Nt in the absence and presence of 1.2 μM PI(4,5)P₂.</p

Generation of potential actin binding mutants in Ysc84 N-terminal YAB domain.

<p>(A) Schematic diagram indicating the site of four conserved pairs of residues selected for mutation. (B) Wild-type and mutant Ysc84 proteins were purified and incubated in the presence or absence of actin during polymerization. Samples were centrifuged at high speed (90K) and supernatant (S) and pellet (P) fractions separated on gels. Data from at least three independent pelleting assays were combined to determine (C) the amount of Ysc84 itself that pellets with F-actin. Error bars are standard deviation; (D) the effect of Ysc84 on the level of actin that is in the supernatant fraction. (E) Pelleting assays were also performed by spinning samples of actin and Ysc84 at low speed (15K) to assess whether the proteins were able to bundle actin filaments. Supernatant and pellet fractions were separated on gels. (F) Data from at least three independent low speed pelleting assays were combined to determine the effect of Ysc84 on the level of actin that is in the pellet fraction. (G)The effect of Ysc84 wild type and the three purified mutant proteins on interaction with actin during polymerization in the presence of actin seeds (0.5 μM G-actin + 0.5 μM F-actin) using a pyrene actin fluorimetry assay. All Ysc84 proteins were added to the reaction at 0.6 μM.</p

Ysc84 and Rvs167 bind to a common site on Las17.

<p>(A) Yeast two-hybrid analysis was carried out in which the polyproline domain of Las17 was carried on an activation plasmid and the Ysc84 or Rvs167 SH3 domains were expressed on the Gal4 binding domain (bait) plasmid. Mutations of Las17 carrying P387A and P388A mutations were used to reveal inhibition of binding of both SH3 domains. (B) Membrane dot blots carrying overlapping 12mer peptides of Las17 over the region 373–406 were incubated with GST fused SH3 domains of Ysc84 and Rvs167 or with GST alone. Proteins were detected by western blotting for GST.</p

Rescue of actin and growth phenotypes of ysc84∆, lsb5(1–142) strain.

<p><i>ysc84∆</i>, <i>lsb5(1–142)</i> strain was transformed with plasmids carrying wild-type or mutant <i>ysc84</i> or with an empty plasmid. (A) Growth on plates was assessed at 30°C and 37°C. (B) Cells were fixed and stained with rhodamine phalloidin before analysing actin organization microscopically. Actin organization in the cells was categorized as detailed.</p

Ysc84 overexpression affects lifetime of different endocytic markers.

<p>(A) The effect of Ysc84 overexpression on endocytosis was assessed by measuring lifetime of different endocytic reporters. Movies of cells expressing fluorescently tagged different endocytic markers transformed with <i>YSC84</i> and an empty plasmid were recorded. At least 30 patches were assessed for each transformant. Error bars are SD. The differences in lifetimes of endocytic markers were calculated using two-tailed Student’s <i>t</i>-test. *** indicates a P value of 0.002 and **** ≥ 0.001. (B) Kymographs were generated from individual patches of fluorescently tagged endocytic markers expressing an empty plasmid or overexpressing Y<i>SC84</i> using the multiple kymographs ImageJ plugin. Arrows mark retractions. (C) Spot tracking of Sla1-GFP patches was performed in cells expressing an empty plasmid or overexpressing <i>YSC84</i> in manual tracking–ImageJ. Red spot–start, green spot–end. The time between spots is 1 second.</p