Significance Membrane asymmetry, curvature, and dynamics have major roles in cellular processes, including vesicle transport. The GTPase ADP ribosylation factor (Arf) and a lipid translocase (flippase) are critical for membrane reorganization during vesicle formation. Direct evidence that Arf and flippase work in concert on membrane transformation/architecture is, however, lacking. We demonstrate that activated Arf-like protein Arl1 interacts with the Arf-activating guanine nucleotide-exchange factor Gea2 and flippase Drs2, forming a ternary complex that is required for lipid asymmetry and Arl1 function at the Golgi. These findings represent a previously missing piece of the puzzle that is our understanding of Arf-mediated membrane remodeling.

In yeast, Arl3p recruits Arl1p GTPase to regulate Golgi function and structure. However, the molecular mechanism involved in regulating activation of Arl1p at the Golgi is unknown. Here, we show that Syt1p promoted activation of Arl1p and recruitment of a golgin protein, Imh1p, to the Golgi. Deletion of SYT1 resulted in the majority of Arl1p being distributed diffusely throughout the cytosol. Overexpression of Syt1p increased Arl1p-GTP production in vivo and the Syt1-Sec7 domain promoted nucleotide exchange on Arl1p in vitro. Syt1p function required the N-terminal region, Sec7 and PH domains. Arl1p, but not Arl3p, interacted with Syt1p. Localization of Syt1p to the Golgi did not require Arl3p. Unlike arl1Δ or arl3Δ mutants, syt1Δ did not show defects in Gas1p transport, cell wall integrity or vacuolar structure. These findings reveal that activation of Arl1p is regulated in part by Syt1p, and imply that Arl1p activation, by using more than one GEF, exerts distinct biological activities at the Golgi compartment.

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.

SummaryGolgins play diverse roles in regulating the structure and function of the Golgi. The yeast golgin Imh1p is targeted to the trans-Golgi network (TGN) through interaction of its GRIP domain with GTP-bound Arl1p. Recycling of Arl1p and Imh1p to the cytosol requires the hydrolysis of GTP bound to Arl1p; however, the point at which GTP hydrolysis occurs remains unknown. Here, we report that selfinteraction of Imh1p plays a role in modulating spatial inactivation of Arl1p. Deletion of IMH1 in yeast decreases the amount of the GTP-bound Arl1p and results in less Arl1p residing on the TGN. Biochemically, purified Imh1p competes with Gcs1p, an Arl1p GTPase-activating protein (GAP), for binding to Arl1p, thus interfering with the GAP activity of Gcs1p toward Arl1p. Furthermore, we demonstrate that the self-interaction of Imh1p attenuates the Gcs1p-dependent GTP hydrolysis of Arl1p. Thus, we propose that the golgin Imh1p serves as a feedback regulator to modulate the GTP hydrolysis of Arl1p.

ADP ribosylation factor (Arf) GTPases are key regulators of membrane traffic at the Golgi complex. In yeast, Arf guanine nucleotide-exchange factor (GEF) Syt1p activates Arf-like protein Arl1p, which was accompanied by accumulation of golgin Imh1p at late Golgi, but whether and how this function of Syt1p is regulated remains unclear. Here, we report that the inositol-requiring kinase 1 (Ire1p)-mediated unfolded protein response (UPR) modulated Arl1p activation at late Golgi. Arl1p activation was dependent on both kinase and endo-RNase activities of Ire1p. Moreover, constitutively active transcription factor Hac1p restored the Golgi localization of Arl1p and Imh1p in IRE1-deleted cells. Elucidating the mechanism of Ire1p–Hac1p axis actions, we found that it regulated phosphorylation of Syt1p, which enhances Arl1p activation, recruitment of Imh1p to the Golgi, and Syt1p interaction with Arl1p. Consistent with these findings, the induction of UPR by tunicamycin treatment increases phosphorylation of Syt1p, resulting in Arl1p activation. Thus, these findings clarify how the UPR influences the roles of Syt1p, Arl1p, and Imh1p in Golgi transport.