Moving and positioning the endolysosomal system

Chemical Immunolog

A membrane-proximal tyrosine-based signal mediates internalization of the HIV-1 envelope glycoprotein via interaction with the AP-2 clathrin adaptor.

The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, mu 2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. The Env cytosolic domain contains two highly conserved tyrosine-based motifs (Y712SPL and Y768HRL), both of which are capable of binding to mu 2 when presented as short peptides. However, only the membrane-proximal motif Y712SPL binds to mu 2 and is required for internalization in the context of the whole cytosolic domain of Env. A glycine residue (Gly711) adjacent to the Y712SPL motif is also important for binding to mu 2/AP-2 and internalization. These observations suggest that the accessibility of the membrane-proximal GY712SPL to mu 2/AP-2 determines its function as a signal for recruitment of HIV-1 Env into clathrin-coated pits and its ensuing internalization

Basolateral sorting of furin in MDCK cells requires a phenylalanine-isoleucine motif together with an acidic amino acid cluster.

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin

Germline mutations in PRKCSH are associated with autosomal dominant polycystic liver disease.

Item does not contain fulltextPolycystic liver disease (PCLD, OMIM 174050) is a dominantly inherited condition characterized by the presence of multiple liver cysts of biliary epithelial origin. Fine mapping established linkage to marker D19S581 (Z(max) = 9.65; theta = 0.01) in four large Dutch families with PCLD. We identified a splice-acceptor site mutation (1138-2A-->G) in PRKCSH in three families, and a splice-donor site mutation (292+1G-->C) in PRKCSH segregated completely with PCLD in another family. The protein encoded by PRKCSH, here named hepatocystin, is predicted to localize to the endoplasmic reticulum. These findings establish germline mutations in PRKCSH as the probable cause of PCLD

Molecular characterization of hepatocystin, the protein that is defective in autosomal dominant polycystic liver disease.

Contains fulltext : 57360.pdf (publisher's version ) (Closed access)BACKGROUND & AIMS: Autosomal dominant polycystic liver disease is characterized by the presence of numerous cysts spread throughout the liver parenchyma. Recently, we discovered that polycystic liver disease is caused by mutations in the protein kinase C substrate 80K-H gene, which encodes a protein named hepatocystin. Previous studies have identified hepatocystin as a protein kinase C substrate, a component of a cytosolic signal transduction complex, a receptor for advanced glycation end products, a vacuolar protein, and the beta subunit of endoplasmic reticulum glucosidase II. Thus, the exact localization and cellular function of hepatocystin remain unclear. METHODS: The localization and biochemical properties of normal and polycystic liver disease mutant forms of hepatocystin were examined by using a combination of immunofluorescence microscopy, immunoblotting, metabolic labeling, immunoprecipitation, and carbohydrate analyses. RESULTS: Normal hepatocystin localizes to the endoplasmic reticulum, where it assembles with the glucosidase II alpha subunit. The 1338-2A-->G truncating mutation in hepatocystin observed in some polycystic liver disease patients produces a protein that is not retained in the endoplasmic reticulum but is secreted into the medium. This mutant protein fails to assemble with the glucosidase II alpha subunit. As a consequence, mutant hepatocystin is undetectable in liver cysts. In addition, levels of normal hepatocystin and of the glucosidase II alpha subunit are substantially reduced in liver and Epstein-Barr virus-immortalized B lymphoblasts from patients with polycystic liver disease. CONCLUSIONS: These findings are consistent with a role of hepatocystin in carbohydrate processing and quality control of newly synthesized glycoproteins in the endoplasmic reticulum. Therefore, altered endoplasmic reticulum processing of some key regulator of cell proliferation may underlie polycystic liver disease

Polycystic liver disease is a disorder of cotranslational protein processing.

Contains fulltext : 47539.pdf (publisher's version ) (Closed access)Autosomal-dominant polycystic liver disease (PCLD) is a rare disorder that is characterized by the progressive development of fluid-filled biliary epithelial cysts in the liver. Positional cloning has identified two genes that are mutated in patients with polycystic liver disease, PRKCSH and SEC63, which encode the beta-subunit of glucosidase II and Sec63, respectively. Both proteins are components of the molecular machinery involved in the translocation, folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum. Most mutations are truncating and probably lead to a complete loss of the corresponding proteins and the defective processing of a key regulator of biliary cell growth. The finding that PCLD is caused by proteins involved in oligosaccharide processing was unexpected and implicates a new avenue for research into neocystogenesis, and might ultimately result in the identification of novel therapeutic drugs

Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA(++)) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA(++) chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA(++) molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HA(wt). These apparently native HA(++) molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane