Identification of a Guanine Nucleotide Exchange Factor for Arf3, the Yeast Orthologue of Mammalian Arf6

Abstract

Small G proteins of the Arf and Rab families are fundamental to the organisation and activity of intracellular membranes. One of the most well characterised of these G proteins is mammalian Arf6, a protein that participates in many cellular processes including endocytosis, actin remodelling and cell adhesion. Exchange of GDP for GTP on Arf6 is performed by a variety of guanine nucleotide exchange factors (GEFs), principally of the cytohesin (PSCD) and EFA6 (PSD) families. In this paper we describe the characterisation of a GEF for the yeast orthologue of Arf6, Arf3, which we have named Yel1 (yeast EFA6-like-1) using yeast genetics, fluorescence microscopy and in vitro nucleotide exchange assays. Yel1 appears structurally related to the EFA6 family of GEFs, having an N-terminal Sec7 domain and C-terminal PH and coiled-coil domains. We find that Yel1 is constitutively targeted to regions of polarised growth in yeast, where it co-localises with Arf3. Moreover the Sec7 domain of Yel1 is required for its membrane targeting and for that of Arf3. Finally we show that the isolated Yel1 Sec7 domain strongly stimulates nucleotide exchange activity specifically on Arf3 in vitro.

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

“…ARF GEFs and ARFs are not highly colocalized (Gillingham and Munro, 2007a;Chun et al, 2008). We found that the majority of Syt1p was colocalized with the late-Golgi and that some Syt1p colocalized with the early Golgi.…”

“…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). Previous studies have also shown that Sec7p and Yel1p are not …”

“…Strong interactions are indicated by additional '+' signs. colocalized with Arl1p (Liu et al, 2005;Gillingham and Munro, 2007a) and are therefore unlikely to be Arl1p-GEFs. Consistent with these findings, our study showed that the Sec7 domain of Yel1p and Sec7p did not interact with Arl1p.…”

“…Despite the fact that the PH domain of yeast Syt1p has poorer homology to the consensus PH domain (Cox et al, 2004), we found that it was necessary for the localization of Syt1p to the Golgi. Unlike Syt1p, Yel1p required both its Sec7 domain and PH domain for targeting to membrane regions of polarized growth in yeast, where it colocalized with Arf3p (Gillingham and Munro, 2007a).…”

“…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).…”