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The epsin‐related adaptor proteins Ent3p and Ent5p participate in budding of clathrin coated vesicles in transport between trans‐Golgi network and endosomes in yeast. Transport of the arginine permease Can1p was analyzed, which recycles between plasma membrane and endosomes and can be targeted to the vacuole for degradation. ent3∆ cells accumulate Can1p‐GFP in endosomes. Can1p‐GFP is transported faster to the vacuole upon induction of degradation in ent5∆ cells than in wild type cells. The C‐terminal domain of Ent5p was sufficient to restore recycling of the secretory SNARE GFP‐Snc1p between plasma membrane and TGN in ent3∆ ent5∆ cells. The SNARE Tlg2p was identified as interaction partner of the Ent5p ENTH domain by in vitro binding assays and the interaction site on Ent5p was mapped. Tlg2p functions in transport from early endosomes to the trans‐Golgi network and in homotypic fusion of these organelles. Tlg2p is partially shifted to denser fractions in sucrose density gradients of organelles from ent5∆ cells while distribution of Kex2p is unaffected demonstrating that Ent5p acts as cargo adaptor for Tlg2p in vivo. Taken together we show that Ent3p and Ent5p have different roles in transport and function as cargo adaptors for distinct SNAREs. [ABSTRACT FROM AUTHOR]

Abstract: Arf GAP proteins are a versatile and diverse group of proteins. They control the activity of the GTP-binding proteins of the ARF family by inducing the hydrolysis of GTP that is bound to Arf proteins. The best-studied role of Arf GAPs is in intracellular traffic. In this review, we will focus mainly on the Arf GAPs that play a role in vesicle formation, Arf GAP1, Arf GAP2 and Arf GAP3 and their yeast homologues, Gcs1p and Glo3p. We discuss the roles of Arf GAPs as regulators and effectors for Arf GTP-binding proteins. [Copyright &y& Elsevier]

The coat protein complex II (COPII) is essential for vesicle formation from the endoplasmic reticulum (ER) and is composed of two heterodimeric subcomplexes, Sec23p/Sec24p and Sec13p/Sec31p, and the small guanosine triphosphatase Sar1p. In an effort to identify novel factors that may participate in COPII vesicle formation, we isolated SMY2, a yeast gene encoding a protein of unknown function, as a multicopy suppressor of the temperature-sensitive sec24-20 mutant. We found that even a low-copy expression of SMY2 was sufficient for the suppression of the sec24-20 phenotypes, and the chromosomal deletion of SMY2 led to a severe growth defect in the sec24-20 background. In addition, SMY2 exhibited genetic interactions with several other genes involved in the ER-to-Golgi transport. Subcellular fractionation analysis showed that Smy2p was a peripheral membrane protein fractionating together with COPII components. However, Smy2p was not loaded onto COPII vesicles generated in vitro. Interestingly, coimmunoprecipitation between Smy2p and the Sec23p/Sec24p subcomplex was specifically observed in sec23-1 and sec24-20 backgrounds, suggesting that this interaction was a prerequisite for the suppression of the sec24-20 phenotypes by overexpression of SMY2. We propose that Smy2p is located on the surface of the ER and facilitates COPII vesicle formation through the interaction with Sec23p/Sec24p subcomplex. [ABSTRACT FROM AUTHOR]

Abstract: In coated vesicle formation, coat protein recruitment needs to be spatially and temporally controlled. The coating process involves conformational changes of the coat protein complexes that activate them for interaction with cargo or machinery components and coat polymerization. Here we discuss mechanisms that have emerged recently from studies of the clathrin adaptor and the COPI systems. [Copyright &y& Elsevier]

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process. [ABSTRACT FROM AUTHOR]

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity. [ABSTRACT FROM AUTHOR]

Syndapins are potential links between the cortical actin cytoskeleton and endocytosis because this family of dynamin-associated proteins can also interact with the Arp2/3 complex activator N-WASP. Here we provide evidence for involvement of N-WASP inter- actions in receptor-mediated endocytosis. We reveal that the observed dominant-negative effects of N-WASP are dependent exclusively on the prolinerich domain, the binding interface of syndapins. Our results therefore suggest that syndapins integrate N-WASP functions in endocytosis. Both proteins co-localize in neuronal cells. Consistent with a crucial rote for syndapins in endocytic uptake, co-overexpression of syndapins rescued the endocytosis block caused by N-WASP. An in vivo reconstitution of the syndapin-N-WASP interaction at cellular membranes triggered local actin polymerization. Depletion of endogenous N-WASP by sequestering it to mitochondria or by introducing anti-N-WASP antibodies impaired endocytosis. Our data suggest that syndapins may act as important coordinators of N-WASP and dynamin functions during the different steps of receptor-mediated endocytosis and that local actin polymerization induced by syndapin-N-WASP interactions may be a mechanism supporting clathrincoated vesicle detachment and movement away from the plasma membrane. [ABSTRACT FROM AUTHOR]