Academic journal article Genetics

The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

Academic journal article Genetics

The Importance of Conserved Features of Yeast Actin-Binding Protein 1 (Abp1p): The Conditional Nature of Essentiality

Article excerpt

ABSTRACT Saccharomyces cerevisiae Actin-Binding Protein 1 (Abp1p) is a member of the Abp1 family of proteins, which are in diverse organisms including fungi, nematodes, flies, and mammals. All proteins in this family possess an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain, a central Proline-Rich Region (PRR), and a C-terminal SH3 domain. In this study, we employed sequence analysis to identify additional conserved features of the family, including sequences rich in proline, glutamic acid, serine, and threonine amino acids (PEST), which are found in all family members examined, and two motifs, Conserved Fungal Motifs 1 and 2 (CFM1 and CFM2), that are conserved in fungi. We also discovered that, similar to its mammalian homologs, Abp1p is phosphorylated in its PRR. This phosphorylation is mediated by the Cdc28p and Pho85p kinases, and it protects Abp1p from proteolysis mediated by the conserved PEST sequences. We provide evidence for an intramolecular interaction between the PRR region and SH3 domain that may be affected by phosphorylation. Although deletion of CFM1 alone caused no detectable phenotype in any genetic backgrounds or conditions tested, deletion of this motif resulted in a significant reduction of growth when it was combined with a deletion of the ADF-H domain. Importantly, this result demonstrates that deletion of highly conserved domains on its own may produce no phenotype unless the domains are assayed in conjunction with deletions of other functionally important elements within the same protein. Detection of this type of intragenic synthetic lethality provides an important approach for understanding the function of individual protein domains or motifs.

SACCHAROMYCES cerevisiae Acting-Binding Protein 1 (Abp1p) was the first described member of a highly conserved family of actin-binding proteins (Drubin et al. 1988) found in diverse organisms including fungi, worms, flies, and humans. The common features of these proteins are an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain (Lappalainen et al. 1998), followed by a large, mainly unstructured central region including a Pro-Rich Region (PRR) and a C-terminal SH3 domain (Figure 1). The conservation among the SH3 domains of these proteins is particularly high (e.g., the human and yeast domains are 45% identical), and they recognize very similar target peptide sequences (Stollar et al. 2009). Given the high conservation and ubiquitous occurrence of Abp1 family members, these proteins undoubtedly fulfill a critical function, and investigating these functions is an important objective. In this work, we have used yeast Abp1p as a model to gain further insight into this family.

Abp1p was originally identified as an actin-binding protein by actin-affinity chromatography (Drubin et al. 1988), and it has been shown to localize to cortical actin patches. Abp1p plays important roles in actin organization and endocytosis. It binds to actin filaments, but not actin monomers, mainly through the ADF-H domain (Lappalainen et al. 1998, Goode et al. 2001), and also possesses two acidic motifs that are required for binding and activation of the Arp2/3 complex (Goode et al. 2001). The SH3 domain mediates biologically relevant interactions with several other proteins involved in endocytosis, such as Ark1p, Scp1p, and Sjl2p (Lila and Drubin 1997; Fazi et al. 2002; Stefan et al. 2005; Haynes et al. 2007; Stollar et al. 2009). The mammalian homolog of Abp1p (mAbp1), similar to the yeast Abp1p, also binds F-actin with its N-terminal actin-binding domain and is involved in receptor-mediated endocytosis (Kessels et al. 2001; Mise-Omata et al. 2003). The SH3 domain mediates protein-protein interactions with proteins involved in synaptogenesis, endocytosis, and cell motility (Kessels et al. 2001; Fenster et al. 2003; Han et al. 2003; Cortesio et al. 2010). mAbp1p is recruited to dynamic actin structures (Kessels et al. 2000), and this localization is reminiscent of the localization of the yeast protein, which is found in cortical actin patches accumulating in the yeast bud but not at actin cables (Drubin et al. …

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