Yeast Arf3p Modulates Plasma Membrane PtdIns(4,5)P2 Levels to Facilitate Endocytosis

Abstract

Phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2] is a key regulator of endocytosis. PtdIns(4,5)P2 generation at the plasma membrane in yeast is mediated by the kinase Mss4p, but the mechanism underlying the temporal and spatial activation of Mss4p to increase formation of PtdIns(4,5)P2 at appropriate sites is not known. Here, we show that ADP ribosylation factor (Arf)3p, the yeast homologue of mammalian Arf6, is necessary for wildtype levels of PtdIns(4,5)P2 at the plasma membrane. Arf3p localizes to dynamic spots at the membrane, and the behaviour of these is consistent with it functioning in concert with endocytic machinery. Localization of Arf3p is disrupted by deletion of genes encoding an ArfGAP homology protein Gts1p and a guanine nucleotide exchange factor Yel1p. Significantly, deletion of arf3 causes a reduction in PtdIns(4,5)P2 at the plasma membrane, while increased levels of active Arf3p, caused by deletion of the GTPase-activating protein Gts1, increase PtdIns(4,5)P2 levels. Furthermore, elevated Arf3p correlates with an increase in the number of endocytic sites. Our data provide evidence for a mechanism in yeast to positively regulate plasma membrane production of PtdIns(4,5)P2 levels and that these changes impact on endocytosis.

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…Similarly, Arf6 stimulates recruitment and activation of type I PIP5 kinases at the plasma membrane and endosomes [6,7], a property shared with its yeast counterpart, yArf3 [8]. At both the Golgi and the plasma membrane, the Arf-dependent recruitment of PtdIns kinases results in enhanced local production of PtdIns(4,5)P 2 .…”

Functions of actin in endocytosis

“…These various data clearly link Arf3 with the endocytic machinery. Analysis of plasma membrane PtdIns(4,5)P2 levels indicate that Arf3 can serve to increase production of this lipid, presumably via the only phosphatidylinositol 4 phosphate 5 kinase in yeast, Mss4 [81]. The production of PtdIns(4,5)P2 is known to be critical for endocytosis as a site for binding the ANTH domain protein Sla2 [32].…”

“…More recently, interactions have also been shown with ArfGEF and ArfGAP proteins. Of these, its guanine exchange factor (GEF) localizes to the plasma membrane in an Arf3-dependent manner [80,81]. The ArfGAP, Gts1 shows extensive overlap in its localization with the endocytic machinery and interacts with many components, including Sla1, Pan1, YAP1801/2 and the clathrin light chain.…”

ADP-ribosylation factor machinery mediates endocytosis in plant cells

“…Similarly, the ARF-GAP VASCU-LAR NETWORK DEFECTIVE 3 (VAN3) controls auxin transport-mediated processes such as vascular tissue formation (9,10), and its cellular role, although not entirely clear, has been mapped to the endosomal compartments (9,11). In contrast to the extensive data in yeast and mammals (4,12,13), very little is known about the direct involvement of the ARF machinery in the endocytosis of plants (14)(15)(16). It has been suggested that the GNOM-like 1 (GNL1) ARF-GEF, the closest homolog of GNOM, regulates the internalization of the PIN2 auxin transporter (17), but its action remained unclear because the loss-offunction gnl1 mutants did not show any endocytosis defects per se (18).…”