Interactions between the Yeast SM22 Homologue Scp1 and Actin Demonstrate the Importance of Actin Bundling in Endocytosis*S⃞

Departments of Molecular Biology and Biotechnology, Biomedical Science, University of Sheffield, Sheffield, S10 2TN, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2008; 283(22):15037-46. DOI: 10.1074/jbc.M710332200
Source: PubMed


The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis,
and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live
cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding
domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent
protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185
to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance
of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis
reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe
a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process.

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Available from: Kathryn R Ayscough, Jan 05, 2016
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    • "Sac6 is the yeast fimbrin homologue and is an actin bundling protein. Its ability to bind actin is necessary for invagination to occur [37], [38], [39]. The bundling of filaments is considered to make a stronger structure to allow invagination to occur against the effect of turgor pressure [30]. "
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    ABSTRACT: Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "These changes are consistent with increases or decreases in the overall actin bundling activity in the cell resulting in decreased or increased actin dynamics respectively. Studies in yeast have revealed similar consequences for the effects of Scp1p, the yeast homologue of SM22, in actin dynamics and actin dependent processes such as endocytosis [11,12,28]. The actin binding and bundling activity of SM22 and related proteins is conferred via sequences c-terminal of the calponin homology (CH) domain including a short linker peptide and one to three calponin-like or 'CLIK23' repeats [29]. "
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    ABSTRACT: SM22 has long been studied as an actin-associated protein. Interestingly, levels of SM22 are often reduced in tumour cell lines, while they are increased during senescence possibly indicating a role for SM22 in cell fate decisions via its interaction with actin. In this study we aimed to determine whether reducing levels of SM22 could actively contribute to a tumourigenic phenotype. We demonstrate that in REF52 fibroblasts, decreased levels of SM22 disrupt normal actin organization leading to changes in the motile behaviour of cells. Interestingly, SM22 depletion also led to an increase in the capacity of cells to spontaneously form podosomes with a concomitant increase in the ability to invade Matrigel. In PC3 prostate epithelial cancer cells by contrast, where SM22 is undetectable, re-expression of SM22 reduced the ability to invade Matrigel. Furthermore SM22 depleted cells also had reduced levels of reactive oxygen species when under serum starvation stress. These findings suggest that depletion of SM22 could contribute to tumourigenic properties of cells. Reduction in SM22 levels would tend to promote cell survival when cells are under stress, such as in a hypoxic tumour environment, and may also contribute to increases in actin dynamics that favour metastatic potential.
    Full-text · Article · Jan 2012 · BMC Cell Biology
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    • "Although some studies have indicated no internalisation defects in cells lacking vps1 expression (Geli and Riezman, 1998), others have demonstrated effects of vps1 deletion on uptake of uracil permease (Fur4) activity (Yu and Cai, 2004). Deletion of many genes encoding proteins with accepted roles in endocytosis, such as Cap1, Scp1, Abp1, are reported to have no defect in several assays (Gheorghe et al., 2008; Huang et al., 1999; Kaksonen et al., 2005; Maldonado-Baez et al., 2008). However, the assays are often performed on fixed cells or over long time courses that do not detect more subtle, kinetic changes in uptake. "
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    ABSTRACT: Dynamins 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.
    Full-text · Article · Oct 2010 · Journal of Cell Science
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