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...