Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2-depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment. INTRODUCTIONLike many viruses, human immunodeficiency virus (HIV)-1 has evolved strategies to avoid detection and destruction by the host. One such strategy is the down-regulation of major histocompatability complex (MHC) class I from the plasma membrane of HIV-1-infected cells, which prevents the cells from being attacked by cytotoxic T lymphocytes. MHC-I down-regulation has been shown to be a function of a virally encoded protein called Nef (Schwartz et al., 1996;Collins et al., 1998). Nef is one of the so-called "accessory proteins" of both human and simian immunodeficiency viruses: although not required for viral replication in vitro, Nef plays a key role in the development of acquired immunodeficiency syndrome. Nef is a small protein, only ϳ200 amino acids, but it has been reported to interact with a large number of host cell proteins, including several proteins that are involved in membrane traffic. These interactions are thought to be responsible for the ability of Nef to modulate the surface expression of MHC-I and other molecules (for review, see Collins and Baltimore, 1999;Piguet et al., 1999;Doms and Trono, 2000;Roeth and Collins, 2006).Among the binding partners that have been identified for Nef are the adaptor protein (AP) complexes. There are four AP complexes in mammalian cells, two of which, AP-1 and AP-2, are highly enriched in clathrin-coated vesicles (CCVs). AP-1 facilitates clathrin-mediated trafficking between the trans-Golgi network (TGN) and endosomes (although there is still some question about directionality), and AP-2 facilitates clathrin-mediated endocytosis (Robinson, 2004). AP-3, which seems to be able to act both in a clathrin-dependent and in a clathrin-independent manner (Dell'Angelica et al., 1998;Peden et al., 2...

Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an $50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an $1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.

The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrinmediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and lowdensity lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats. Cargo proteins are sorted and packaged into clathrincoated vesicles (CCVs) by interacting with adaptors, which bind to specific sorting signals in the cytoplasmic tails of the cargo proteins and link them to clathrin. There are two highly abundant adaptors in mammalian CCVs: AP-1 (for adaptor protein complex 1), which is involved in clathrin-mediated intracellular trafficking, and AP-2, which is involved in clathrin-mediated endocytosis. Both AP-1 and AP-2 are heterotetramers, consisting of two large subunits (g and b1 in AP-1, a and b2 in AP-2), a mediumsized (m) subunit and a small (s) subunit (1,2). The m subunits bind to sorting signals with the consensus sequence YXXF (3,4), and the g-type large subunits together with the s subunits bind to sorting signals with the consensus sequence [D/E]XXXL[L/I] (5). Clathrin-coated vesicle preparations from different tissues contain different ratios of AP-1 to AP-2 (e.g. in mammary gland, there is more AP-1, whereas in brain there is more AP-2) (6), but invariably, the AP complexes are major components of the CCVs, second in abundance only to clathrin itself.Over the last 2 years, others and we have been investigating the functions of the AP-1 and AP-2 complexes in tissue culture cells using siRNA knockdowns. Surprisingly, we have found that even when we deplete AP-2 to undetectable levels, some cargo proteins are still endocytosed normally in a clathrin-dependent manner. Thus, the transferrin receptor, which has a YX...