Distinct role of TGN-resident clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.

Clathrin-mediated vesicle trafficking plays central roles in post-Golgi transport. In yeast (Saccharomyces cerevisiae), the AP-1 complex and GGA adaptors are predicted to generate distinct transport vesicles at the trans-Golgi network (TGN), and the epsin-related proteins Ent3p and Ent5p (collectively Ent3p/5p) act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGA adaptors are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here, we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We show that AP-1 disruption in the ent3Δ5Δ mutant impaired transport of the Vps21p guanine nucleotide exchange factor Vps9p transport to the Vps21p compartment and severely reduced Vps21p activity. Additionally, GGA adaptors, the phosphatidylinositol-4-kinase Pik1p and Rab11 GTPases Ypt31p and Ypt32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.

Distinct roles for plasma membrane PtdIns(4)P and PtdIns(4,5)P2 during receptor-mediated endocytosis in yeast.

Clathrin-mediated endocytosis requires the coordinated assembly of various endocytic proteins and lipids at the plasma membrane. Accumulating evidence demonstrates a crucial role for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] in endocytosis but specific roles for phosphatidylinositol-4-phosphate [PtdIns(4)P], other than as the biosynthetic precursor of PtdIns(4,5)P2, have not been clarified. In this study we investigated the roles of PtdIns(4)P and PtdIns(4,5)P2 in receptor-mediated endocytosis through the construction of temperature-sensitive (ts) mutants for the phosphatidylinositol 4-kinases (PI4-kinases) Stt4p and Pik1p and the 1-phosphatidylinositol-4-phosphate 5-kinase [PtdIns(4) 5-kinase] Mss4p. Quantitative analyses of endocytosis revealed that both the stt4tspik1ts and mss4ts mutants have a severe defect in endocytic internalization. Live-cell imaging of endocytic protein dynamics in stt4tspik1ts and mss4ts mutants revealed that PtdIns(4)P is required for the recruitment of the α-factor receptor Ste2p to clathrin-coated pits, whereas PtdIns(4,5)P2 is required for membrane internalization. We also found that the localization to endocytic sites of the ENTH/ANTH domain-bearing clathrin adaptors, Ent1p, Ent2p, Yap1801p and Yap1802p, is significantly impaired in the stt4tspik1ts mutant but not in the mss4ts mutant. These results suggest distinct roles in successive steps for PtdIns(4)P and PtdIns(4,5)P2 during receptor-mediated endocytosis.

Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis.

The dynamic assembly and disassembly of actin filaments is essential for the formation and transport of vesicles during endocytosis. In yeast, two types of actin structures, namely cortical patches and cytoplasmic cables, play a direct role in endocytosis, but how their interaction is regulated remains unclear. Here, we show that Srv2/CAP, an evolutionarily conserved actin regulator, is required for efficient endocytosis owing to its role in the formation of the actin patches that aid initial vesicle invagination and of the actin cables that these move along. Deletion of the SRV2 gene resulted in the appearance of aberrant fragmented actin cables that frequently moved past actin patches, the sites of endocytosis. We find that the C-terminal CARP domain of Srv2p is vitally important for the proper assembly of actin patches and cables; we also demonstrate that the N-terminal helical folded domain of Srv2 is required for its localization to actin patches, specifically to the ADP-actin rich region through an interaction with cofilin. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin cytoskeleton during clathrin-mediated endocytosis.

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins.

Rab5-mediated endosome formation is regulated at the trans-Golgi network.

Early endosomes, also called sorting endosomes, are known to mature into late endosomes via the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence is thought to be maintained by the continual fusion of transport vesicles from the plasma membrane and the trans-Golgi network (TGN). Here we show instead that endocytosis is dispensable and post-Golgi vesicle transport is crucial for the formation of endosomes and the subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all three proteins required for endosomal nucleotide exchange on Vps21p are first recruited to the TGN before transport to the endosome, namely the GEF Vps9p and the epsin-related adaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, with Vps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These results provide a different view of endosome formation and identify the TGN as a critical location for regulating progress through the endolysosomal trafficking pathway.

Yeast Eps15-like endocytic protein Pan1p regulates the interaction between endocytic vesicles, endosomes and the actin cytoskeleton.

The actin cytoskeleton plays important roles in the formation and internalization of endocytic vesicles. In yeast, endocytic vesicles move towards early endosomes along actin cables, however, the molecular machinery regulating interaction between endocytic vesicles and actin cables is poorly understood. The Eps15-like protein Pan1p plays a key role in actin-mediated endocytosis and is negatively regulated by Ark1 and Prk1 kinases. Here we show that pan1 mutated to prevent phosphorylation at all 18 threonines, pan1-18TA, displayed almost the same endocytic defect as ark1Δ prk1Δ cells, and contained abnormal actin concentrations including several endocytic compartments. Early endosomes were highly localized in the actin concentrations and displayed movement along actin cables. The dephosphorylated form of Pan1p also caused stable associations between endocytic vesicles and actin cables, and between endocytic vesicles and endosomes. Thus Pan1 phosphorylation is part of a novel mechanism that regulates endocytic compartment interactions with each other and with actin cables.

Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole.

The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

Peroxiredoxin stabilization of DE-cadherin promotes primordial germ cell adhesion.

Regulated adhesion between cells and their environment is critical for normal cell migration. We have identified mutations in a gene encoding the Drosophila hydrogen peroxide (H2O2)-degrading enzyme Jafrac1, which lead to germ cell adhesion defects. During gastrulation, primordial germ cells (PGCs) associate tightly with the invaginating midgut primordium as it enters the embryo; however, in embryos from jafrac1 mutant mothers this association is disrupted, leaving some PGCs trailing on the outside of the embryo. We observed similar phenotypes in embryos from DE-cadherin/shotgun (shg) mutant mothers and were able to rescue the jafrac1 phenotype by increasing DE-cadherin levels. This and our biochemical evidence strongly suggest that Jafrac1-mediated reduction of H2O2 is required to maintain DE-cadherin protein levels in the early embryo. Our results present in vivo evidence of a peroxiredoxin regulating DE-cadherin-mediated adhesion.