Characterization for the function of yeast Arf3p and rf3p-interacting proteins

ADP-ribosylation factors (ARFs) are ubiquitous regulators of vesicular membrane traffic. Yeast Arf3p, which is most similar to mammalian ARF6 is not required for endocytosis but involves in polarity development and actin organization. In this study, the role of Arf3p has been further characterized, and it has been shown that Arf3p is required for not merely filamentous growth in galactose-containing medium but also invasive growth on rich medium plate. Moderate expression of either Arf3-GFP or non-tagged integrated Arf3p could partially rescue the filamentous defect observed in arf3 mutant. However, the invasive defect of arf3 could not be rescued by any ARF3 constructs. In addition, the invasive defect could be fully restored in diploid heterozygous arf3 mutant containing wild-type allele of ARF3. Recent study for Arf3p has shown Yel1p (Yeast EFA6-like-1) acts as one of the guanine nucleotide exchange factors (GEFs) for Arf3p. The physical interaction between Arf3p and Yel1p was further confirmed by yeast two-hybrid analysis in this study. Moreover, another Arf3p-interacting protein, Afi1p (Arf3p-interacting protein 1) has been identified in our previous study. Further examination of the two Arf3p-interacting proteins, Yel1p and Afi1p, revealed that Afi1p was required for both filamentous and invasive growth; but Yel1p only involves in invasiveness of yeast cells. Integrated form of Arf3p-interacting Afi1p could partially rescue the filamentous defect in afi1. N-terminal mutant of Afi1p (Afi138KLGP4A) which did not interact with Arf3p could not rescue the filamentous defect in afi1. In this filamentous strain of Σ1278b background, arf3, afi1, or yel1 deletion mutants exhibited defect on actin patch polarization, consistent with previous studies. To further determine the function of Afi1p, C-terminal truncation of Afi1p revealed that lacking of C-terminal 20 amino acids did not affect either Afi1p itself polarization or Arf3p polarization but actin patches could not polarize properly. In short, this study demonstrated that Arf3p and Afi1p are both indispensable for filamentous and invasive growth, but Yel1p is not. Arf3p and its interacting proteins, Afi1p and Yel1p, all involve in regulation for actin cytoskeleton organization. From this study, N-terminal Afi1p which is critical for interacting with Arf3p may act in the same pathway with Arf3p, while C-terminal Afi1p may have other functions than maintaining Arf3p polarization. The detailed mechanism of Arf3p and Afi1p to regulate filamentous and invasive growth requires further investigation.