Afi1p Functions as an Arf3p Polarization-specific Docking Factor for Development of Polarity

Abstract

ADP-ribosylation factors (Arfs) are highly conserved small GTPases and are critical components of vesicle trafficking. Yeast Arf3p, despite its similarity to mammalian Arf6, is not required for endocytosis but is involved in polarity development. In this study, we identified an Arf3p interacting protein 1 (Afi1p), which, through its N-terminal conserved region, specifically interacts with GTP-bound Arf3p. Afi1p is distributed asymmetrically at the plasma membrane and is required for polarized distribution of Arf3p but not of an Arf3p guanine nucleotide-exchange factor, Yel1p. However, Afi1p is not required for targeting of Arf3p or Yel1p to the plasma membrane. Like arf3 mutant yeast, afi1 mutant yeast exhibited an abnormal budding pattern and partially delayed actin patch polarization. An Afi1p, 38 KLGP4A-Afi1p, mutated at the Arf3p-binding region, loses its ability to interact with Arf3p and maintain the polarized distribution of Arf3p. Although 38 KLGP4A-Afi1p still possessed a proper polarized distribution, it lost its ability to rescue actin patch polarization in afi1 mutant cells. Our findings demonstrate that Afi1p functions as an Arf3p polarization-specific adapter and participates in development of polarity.

Syt1p promotes activation of Arl1p at the late Golgi to recruit Imh1p

“…Yeast cells contain five structurally related Sec7 domain-containing proteins with the potential to provide GEF activity for ARF family proteins involved in vesicular transport and actin remodeling (Casanova, 2007;Anders and Jurgens, 2008;Tsai et al, 2008). Sec7p, Gea1p, Gea2p and Syt1p have been shown to be Arf1/2 GEFs and Yel1p acts as an Arf3p-GEF (Jones et al, 1999;Gillingham and Munro, 2007a;Gillingham and Munro, 2007b).…”

“…Studies using a temperature-sensitive GEA1 or GEA2 mutant suggest that they are involved in ER-Golgi and intra-Golgi transport (Peyroche et al, 2001). Yel1p acts as a GEF for Arf3p and is involved in Arf3p-dependent actin-patch polarization (Gillingham and Munro, 2007a;Tsai et al, 2008). Syt1p acts as a GEF for Arf2p in vitro and could be involved in secretion (Jones et al, 1999).…”

Arf3p GTPase is a key regulator of Bud2p activation for invasive growth inSaccharomyces cerevisiae

“…We observed that although Arf3p L23V –green fluorescent protein (GFP) and Arf3p I33V -GFP exhibited similar subcellular distributions compared with wild-type Arf3p based on microscopic observations and fractionation analysis (Supplemental Figure S2, C and D), coimmunoprecipitation analysis revealed that the interaction between Arf3p I33V and HA-Bud2p was weakened (Figure 2I). Of interest, neither L23 nor I33 of Arf3p was required for interaction with its GEF, Yel1p (Gillingham and Munro, 2007), or its polarization partner, Afi1p (Tsai et al , 2008), in yeast two-hybrid analysis (Supplemental Figure S2E). These data indicate that I33 of Arf3p specifically contributes to the Arf3p–Bud2p interaction.…”

“…To examine whether Arf3p activity could be affected by glucose depletion, we measured the amount of active Arf3p using recombinant GST-Afi1N to pull down Arf3p-GTP (Tsai et al , 2008). GST-Afi1N specifically bound to active Arf3p Q71L but not inactive Arf3p T31N (Figure 8A).…”

Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases

“…Interestingly, a GATOR1 complex, which is also composed of two DENN-related proteins (NPRL2 and NPRL3) and DEPDC5, has been reported to exhibit GAP activity toward RagA/B, even though the DENN-related domains of both NPRL2 and NPRL3 contain a u-DENN (longin) domain alone and lack c-DENN and d-DENN domains Bar-Peled et al, 2013). The yeast (Saccharomyces cerevisiae) protein Afi1p has also been identified as a DENN-related protein Levine et al, 2013a) that specifically binds GTP-bound Arf3p, but no Afi1p homologues have ever been identified in animals (Tsai et al, 2008). Based on these findings together with the fact that sequence conservation between classical DENN and DENN-related domains is very low, DENNrelated proteins are likely to function as regulators of other small GTPases, e.g., Rags and Arfs, rather than as RabGEFs.…”