Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrp1p), known membrane traffic regulators (Kes1p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting.exocytosis ͉ lipid rafts ͉ Saccharomyces cerevisiae ͉ sorting ͉ Golgi T he mechanisms responsible for sorting proteins to the cell surface from the Golgi complex are poorly understood in eukaryotic cells. The trans-Golgi network (TGN) has been recognized as a major hub for sorting (1). However, there is also evidence that sorting occurs in endosomes (2). In polarized cells such as epithelial cells and neurons, biosynthetic cargo is delivered to separate membrane domains by pathways employing different sorting principles (3,4). Recent work has demonstrated that yeast cells also have at least two separate routes to the cell surface (5-8). Little is known about the genes that are responsible for sorting and packaging surface cargo into different transport containers. Previous screens aimed at identifying this machinery relied, for example, on major growth defects and the internal accumulation of invertase, which has been later shown to be transported by the minor pathway to the plasma membrane (7,8). These screens have mainly identified mutants that blocked endoplasmic reticulum (ER)-to-Golgi transport and delivery to the plasma membrane (9, 10). However, mutations in regulators of post-Golgi sorting and vesicle formation with few exceptions have not been detected by such screens, probably because a block in one transport route to the cell surface can be rescued by partial rerouting from the affected to the undisturbed pathway (7,8).Here we describe a visual screening procedure devised to circumvent this problem. We aimed at developing an assay sensitive enough to detect sorting defects within the secretory pathway and applicable to genome-wide screening. The screen takes advantage of the systematic yeast knockout array (11), which should contain the nonessential genes responsible for regulating cargo entry into specialized, partially redundant pathways. The results of this genome-wide screen demonstrate the suitability of our visual screening approach for identifying regulators of sorting and vesicle formation involved in surface delivery of biosynthetic cargo. Table 1. Images (GFP and DIC) ...

Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.