Abstract
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.
Highlights
Recent studies have given us a framework to begin to understand the process of endocytosis
Distinct stages of endocytosis have been determined, the role of many of the proteins that localize to the cortical complexes is largely unknown
Cells that have a disassembled actin cytoskeleton due to treatment with the drug latrunculin-A or that have a stabilized actin cytoskeleton that cannot disassemble after jasplakinolide addition cannot perform endocytosis [19, 28]
Summary
Yeast Strains and Cell Growth—Yeast strains used in this study are listed in supplemental Table 1. Transformations were performed using lithium acetate as previously described [11]. Overexpression was carried out using previously generated plasmids pKA280 and pKA281 that were transformed in wild-type and act102 strains [5]. Mutagenesis was carried out using the Stratagene QuikChange kit according to the manufacturer’s instructions and using oligonucleotides listed in supplemental Table 3. Protein Purification and Assays—Scp was purified as described previously [5] by expression of SCP1 from pKA211 in Escherichia coli BL21(DE3) cells. Yeast actin was purified as described [13]. Rabbit skeletal muscle actin was purified as described previously [14]. Cells expressing tagged proteins were visualized after growing to early log phase in synthetic medium with appropriate supplements.
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