Characterisation of SCP1P, an actin-bundling protein in the budding yeast Saccharomyces cerevisiae

The organisation of the actin cytoskeleton within eukaryotic cells is controlled by a large variety of actin-binding proteins (ABPs) with roles ranging from nucleation to bundling, severing and capping of actin filaments. The calponins and transgelins are a family of actin-binding/bundling proteins possessing one or more highly conserved motifs (i) calponin homology (CH) domain, and (ii) calponin-like repeat (CLR/ CLIK23 repeat), which have both been implicated in acting-binding. The Saccharomyces cerevisiae gene SCP1 encodes a protein, which shares a high degree of homology with SM22/transgelin. In this study Scp1p was purified and, using a range of biochemical techniques was shown to possess actin-bundling activity. Further analysis by EM revealed the formation of tight bundles of actin in the presence of Scp1p. Although a large number of protein containing CH domains have been shown to bind actin, the CH domain alone is not sufficient to bind actin. Using amino and carboxy terminal (N- and C-term) Scp1p truncated mutants it has been possible to localise the actin-binding domain (ABD) to the C-terminal region containing the CLR/CLIK23 repeat, and to show that the CH domain alone is not able to bind actin. In vivo studies into the localisation of Scp1p have revealed a co-localisation with actin to patches at the cell cortex. Other proteins found at these actin patches include the yeast fimbrin homologue Sac6p, another CH domain-containing protein involved in actin bundling. It has been shown that deletion of the SCP1 gene alone has no adverse affect on actin organisation, whereas a SAC6/SCP1 double knockout displays a more defective actin phenotype than the SAC6 single knockout. The importance of Scp1p in organising the actin cytoskeleton is clearly shown in cells overexpressing Scp1p, which possess a severely disrupted actin cytoskeleton, with the appearance of large clumpy actin patches. Taken together this study shows that Scp1p is an actin-bundling protein, which is involved with Sac6p in the organisation of the yeast cytoskeleton