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

Regulation of Actin Dynamics by Ysc84 and Las17 in Yeast Endocytosis

Regulation of Actin Dynamics by Ysc84 and Las17 in Yeast Endocytosis This study has focused on the role of a protein Ysc84 (mammalian homologue SH3yl-1). Previously we demonstrated that Ysc84 binds and bundles F-actin. Furthermore, this binding occurs only when Ysc84 is bound by the Arp2/3 activator Las17 (homologue of WASP). In vivo Ysc84 localizes to endocytic patches prior to invagination and disassembles after scission. However, deletion of ysc84 in vivo only has subtle effect on endocytosis. Furthermore, a double deletion of ysc84 and its highly related homologue lsb3 did not add significantly to its endocytic phenotype. Over 60 proteins have been identified to function in yeast endocytosis and many of these have mammalian homologues also found at endocytic sites. In yeast, the actin cytoskeleton plays a critical role in generating the force required to drive invagination and many actin binding proteins function to regulate actin during this process. While many actin-binding proteins are known, the mechanism of actin regulation during endocytosis is not well understood. This project is aimed at increasing our understanding the function of Ysc84 in yeast endocytosis and how Ysc84 and Las17 function together during this complex process. Specifically we have investigated whether overexpression of YSC84 affects endocytosis in vivo. We have also used mutagenesis to identify key residues involved in actin binding and regulation of Ysc84 through both biochemical and live cell imaging approaches. In addition the importance of newly identified actin bind site within Las17 polyproline region was investigated in vivo. This study showed that Ysc84 overexpression affects invagination and scission stages of endocytosis. The involvement of Ysc84 in maintaining the vacuolar morphology was also discovered. At least one actin binding site on Ysc84 was mapped and determined in vitro, and the consequence of impaired actin binding by Ysc84 was investigated in vivo. Ysc84 was also found to bind a range of lipids and to be phosphorylated. This study also showed a novel role of the polyproline region of Las17 in actin binding and that it is differently involved in regulation of endocytosis in vivo compared to the WH2 domain.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Function and interactions of the Ysc84/SH3yl1 family of actin- and lipid-binding proteins

Understanding how actin filaments are nucleated, polymerized and disassembled in close proximity to cell membranes is an area of growing interest. Protrusion of the plasma membrane is required for cell motility, whereas inward curvature or invagination is required for endocytic events. These morphological changes in membrane are often associated with rearrangements of actin, but how the many actin-binding proteins of eukaryotes function in a co-ordinated way to generate the required responses is still not well understood. Identification and analysis of proteins that function at the interface between the plasma membrane and actin-regulatory networks is central to increasing our knowledge of the mechanisms required to transduce the force of actin polymerization to changes in membrane morphology. The Ysc84/SH3yl1 proteins have not been extensively studied, but work in both yeast and mammalian cells indicate that these proteins function at the hub of networks integrating regulation of filamentous actin (F-actin) with changes in membrane morphology

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

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

The effect of the Las17 RRRR-AAAA mutation on recruitment of key endocytic proteins.

<p>Strains expressing reporters for early stages of endocytosis (Sla1-GFP) and for Arp2/3 (Arc15-mCherry) were generated. The behaviour of reporters was analysed in otherwise wild-type cells (A) or in cells harbouring an integrated mutant allele of Las17 (<i>las17 RRRR-AAAA</i>) (B). The profiles show fluorescence intensity over time for 3 different patches analysed. A total of 30 patches were analysed for each strain and the combined data are shown in (C). Error bars denote standard deviation. Sla1-GFP has a significantly different lifetime between the strains (**** students t-test p<0.0001). Arc15-mCherry has a significantly different lifetime between the strains (*** students t-test p = 0.0009). Mean lifetimes are given. n = 30.</p