Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyperor hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/ lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extraand intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease.

In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to "tether" vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimidesensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function. INTRODUCTIONSoluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are central to membrane fusion, forming tight 4 helix bundles that pull opposing membranes together (Hay, 2001). Although much is known about how SNARE complexes form, less is known about the upstream processes that regulate SNARE assembly. Some specificity is contributed by the SNARE proteins themselves, which catalyze fusion only in particular combinations due to the conformation of both the SNARE domain and N-terminal regulatory domain (Sollner, 2003). However, this intrinsic specificity is unlikely to suffice in vivo, and many other SNARE-interacting proteins have been proposed to contribute to the specificity of membrane fusion (Wickner and Haas, 2000;Chen and Scheller, 2001; Hey, 2001;Wendler and Tooze, 2001;Sollner, 2003).One important function that controls fusion specificity is the aggregation of compartments before the formation of trans-SNARE pairs or SNARE-pins. So-called "tethering" factors provide this function and are typically comprised of oligomeric complexes that associate directly or indirectly with SNARE proteins, Rab proteins, and Sec1/Munc-like (S/M) proteins (Waters and Pfeffer, 1999;Whyte and Munro, 2002). Only limited sequence or mechanistic conservation has been observed between tethering complexes, implying that each complex is tailored to provide and integrate ...

These authors contributed equally to this work.Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell. HIV-1 infection is characterized by a lengthy latent period before the onset of acquired immunodeficiency syndrome (AIDS). During this period, abundant viral production is kept in check by the immune system and cells that are killed by infection are replaced. Despite mounting a strong early immune response, HIV-1 expression progressively depletes CD4þ T cells, a situation that leads to a progressive weakening of the immune response to infection and the onset of AIDS (1,2). HIV-1 gene transcription generates a primary 9-kilobase pair (kbp) RNA that has three fates dictated by a tight regulatory circuit and temporal activities of viral proteins. The 9-kbp RNA is multiply spliced following transcription to generate several 2-kbp RNAs that give rise to regulatory proteins Tat, Rev and Nef. Tat accumulates and is primarily responsible for high-level transactivation of the integrated HIV-1 provirus. Once a threshold level of Rev is reached, a molecular switch occurs to promote the inhibition of splicing of the primary transcript. The decreased splicing activity also produces singlyspliced RNA species (4-kbp) (3). Rev binds the Revresponsive cis-acting element RNA (4) to promote the nuclear export of the 9-kbp and singly-spliced 4-kbp HIV-1 RNAs. The 9-kbp RNA is not only a substrate for the translation machinery to generate structural (Gag) and viral enzymes, but in addition, it is selected for encapsidati...

Through a proteomic analysis of clathrin-coated vesicles from rat liver we identified the mammalian homolog of receptor-mediated endocytosis 8 (RME-8), a DnaJ domain-containing protein originally identified in a screen for endocytic defects in Caenorhabditis elegans. Mammalian RME-8 has a broad tissue distribution, and affinity selection assays reveal the ubiquitous chaperone Hsc70, which regulates protein conformation at diverse membrane sites as the major binding partner for its DnaJ domain. RME-8 is tightly associated with microsomal membranes and co-localizes with markers of the endosomal system. Small interfering RNA-mediated knock down of RME-8 has no influence on transferrin endocytosis but causes a reduction in epidermal growth factor internalization. Interestingly, and consistent with a localization to endosomes, knock down of RME-8 also leads to alterations in the trafficking of the cation-independent mannose 6-phosphate receptor and improper sorting of the lysosomal hydrolase cathepsin D. Our data demonstrate that RME-8 functions in intracellular trafficking and provides the first evidence of a functional role for a DnaJ domainbearing co-chaperone on endosomes.

Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-2-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Clathrin-coated vesicle formation represents the initial step of the major pathway for receptor-mediated endocytosis. The known roles of AP-2, 1 clathrin and dynamin in this process are as follows. AP-2 is believed to drive clathrin assembly at the plasma membrane and binds to tyrosine-based internalization signals, playing a central role in both formation and function of clathrin-coated pits (CCPs); clathrin gives an organizing framework to the pit; and the dynamin GTPase activity is required for membrane fusion events leading to coated vesicle formation (1-5).The mechanisms by which these soluble proteins are targeted to the plasma membrane to form an organized clathrin coat have been extensively studied. Targeting of AP-2 complexes is mediated by its ␣-adaptin subunit. Studies of chimeras in which equivalent domains of ␣-adaptin from AP-2 and ␥-adaptin from AP-1 were exchanged allowed the identification of a CCP targeting signal within a 200-amino acid region of the N-terminal "head" or "trunk" domain of ␣-adaptin. These studies also established a role for the C-terminal "ear" domain of ␣-adaptin (6, 7), a result confirmed by recent data showing efficient targeting of a GFP-ear construct to CCP (8). Furthermore, the ␣-adaptin N-terminal domain also contains a phosphoinositide binding site (9), which seems necessary for efficient incorporation of AP-2 complexes into CCPs (10). Clathrin assembly at the plasma membrane requires plasma membrane-bound AP-2 complexes (11,12) and is thought to result from direct AP-2/clathrin interactions. Dynamin directly binds to salt-stripped membranes and to phospholipids containing liposomes (13-15), suggesting that dynamin might directly interact with the plasma membrane in vivo. This hypothesis was confirmed by the fact that dynamin is homogeneously redistributed at the plasma membrane when CCP assembly is inhibited (8). Its localization to CCPs requires its C-terminal proline-rich domain (16), which binds to the SH3 domain of amphiphysin (17). Recently, several new CCP-associated proteins were identified including Eps15 (18), amphiphysin (19,20), and epsin (21). The mechanisms by which these accessory coat proteins are targeted to CCPs have not yet been investigated.The Eps15 protein is constitutively and ubiquitously associated with AP-2 (22). Recent data obtained both in vivo and in a perforated cell assay showed that Eps15 is required for the early steps of clathrin-dependent endocytosis (23,24). The fact that Eps15 is not found in coated vesicles (25) suggested a coated pit-restricted function. Results showing that the ␣...

endocytosis ͉ huntingtin-interacting protein ͉ trans-Golgi network

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca(2+) concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.

Delivery of endocytosed macromolecules to mammalian cell lysosomes occurs by direct fusion of late endosomes with lysosomes, resulting in the formation of hybrid organelles from which lysosomes are reformed. The molecular mechanisms of this fusion are analogous to those of homotypic vacuole fusion in Saccharomyces cerevisiae. We report herein the major roles of the mammalian homolog of yeast Vps18p (mVps18p), a member of the homotypic fusion and vacuole protein sorting complex. When overexpressed, mVps18p caused the clustering of late endosomes/lysosomes and the recruitment of other mammalian homologs of the homotypic fusion and vacuole protein sorting complex, plus Rab7-interacting lysosomal protein. The clusters were surrounded by components of the actin cytoskeleton, including actin, ezrin, and specific unconventional myosins. Overexpression of mVps18p also overcame the effect of wortmannin treatment, which inhibits membrane traffic out of late endocytic organelles and causes their swelling. Reduction of mVps18p by RNA interference caused lysosomes to disperse away from their juxtanuclear location. Thus, mVps18p plays a critical role in endosome/lysosome tethering, fusion, intracellular localization and in the reformation of lysosomes from hybrid organelles.