Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26⌬ mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP when immunoprecipitated from detergent-solublized ER membranes. Based on these observations, we propose that Erv26p serves as a transmembrane adaptor to link specific secretory cargo to the COPII coat. Because ALP is a type II integral membrane protein in yeast, these findings imply that an additional class of secretory cargo relies on adaptor proteins for efficient export from the ER. INTRODUCTIONThe eukaryotic secretory pathway transports a remarkable variety of cargo proteins to their proper cellular location. Several lines of evidence indicate that targeting information encoded within a secretory protein is recognized by the intracellular transport machinery to direct localization. Cytosolic coat protein complexes play an important role in deciphering targeting information and act in the selection of secretory proteins into appropriate transport intermediates (Bonifacino and Glick, 2004). However, the sorting signals and mechanisms by which coat complexes accommodate such diversity in secretory cargo remain poorly understood.Protein transport from the endoplasmic reticulum (ER) relies on coat protein complex II (COPII), which selects fully folded secretory cargo into ER-derived transport intermediates. COPII coats consist of the small GTPase Sar1p, the Sec23/24 complex, and the Sec13/31 complex (Barlowe et al., 1994). Biochemical and structural studies have demonstrated that the Sec24p subunit of this coat complex contains multiple cargo recognition sites and binds specific sorting signals displayed on the cytosolic regions of secretory proteins (Miller et al., 2003;Mossessova et al., 2003). Diacidic sequences and other polypeptide sorting signals have been identified in cargo proteins that interact with amino acid residues within defined Sec24p cargo recognition sites. Current models envisage that the cargo recognition and binding by Sec24p is stabilized through assembly of prebudding complexes consisting of Sec23/24 and Sar1p-GTP bound to secretory cargo on the ER membrane surface. These prebudding complexes are then incorporated into an outer layer Sec13/31 scaffold structure that deforms the membrane and produces COPII-coated vesicles (Lee et al., 2004;Stagg et al., 2006).In addition to the COPII coat proteins, cargo-specific accessory factors are required to accommodate the variety of secretory cargo exported from the ER in CO...

Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.The early endocytic pathway is increasingly being recognized as a complex and heterogeneous membrane population in which distinct endosomal populations are specialized for the trafficking of different receptor types (1, 2). Complexity and specialization in the endosomal pathway are achieved by the action of small GTPases and by the generation of specific phosphoinositides on the endosomal surface. One of the best studied examples of this mechanism is the specific and temporal targeting of proteins containing FYVE domains to phosphatidylinositol 3-phosphate (3-6), which is present almost exclusively in endosomal membranes. The human genome encodes for Ͼ30 proteins that contain FYVE domains, several of which are highly conserved and which may contribute in different ways toward establishing the complexity and functionality of the endocytic pathway. We recently characterized one of these proteins, WDFY2, named for its content of WD40 motifs and a FYVE domain (7). In Caenorhabditis elegans, WDFY2 depletion impairs endocytosis in coelomocytes, and in mammalian cells it defines a distinct set of endosomes that lack the canonical markers EEA1 and Rab5 and are further distinguished by their close proximity to the plasma membrane (7,8).In addition to internalization, the endosomal pathway plays a critical role in modulating signal transduction. Growth factor receptors are internalized immediately following activation, and both their fate and their signaling functions are affected by their transit through the endocytic pathway (9 -13). Different receptors traffic through distinct early endosomal compartments (1, 2), and their signaling functions are modulated by the specific nature of the endosomes through which they traffic. For example, signaling by transforming growth factor ␤ is influenced by the endosomal localization of the SMAD-interacting protein SARA, which is found in endosomes containing the...

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Nascent proteins translated and processed in the endoplasmic reticulum (ER) sometimes contain intrinsic signals for ER retention or ER retrieval. These signals are usually a few amino acids in length, and if alanine modifications are made within these sequences, normal transit patterns of the nascent protein frequently change. The purpose of this study was to determine whether two alanines juxtaposed to the first globular domain of aggrecan's core protein affect its transit in Chinese hamster ovary (CHO) cells. Results show that two alanines juxtaposed to the first globular domain (G1AA) minimized secretion of the protein. However, transgenic proteins with juxtaposed glutamate-phenylalanine (G1EF) or no additional amino acids (G1) were still secreted. GFP-tagged G1AA localized in the lumen of the ER but not in the Golgi. In contrast, a portion of GFP-tagged G1EF and G1 did appear in the Golgi compartment. More importantly, unique and striking accumulations of G1EF and G1 transgenic proteins were seen in large dilated regions of the ER cisternae, reminiscent of accumulations seen in alpha1-antitrypsin deficiency disease. G1AA transgenic proteins did not form these vesicles but were diffusely distributed throughout the ER lumen. These results indicate that just two juxtaposed alanines can profoundly affect a large globular protein's intracellular localization.

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