Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes.

The Nef protein of primate lentiviruses down-regulates the cell surface expression of CD4 and probably MHC I by connecting these receptors with the endocytic machinery. Here, we reveal that Nef interacts with the mu chains of adaptor complexes, key components of clathrin-coated pits. For human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) Nef, this interaction occurs via tyrosine-based motifs reminiscent of endocytosis signals. Mutating these motifs prevents the binding of SIV Nef to the mu chain of plasma membrane adaptor complexes, abrogates its ability to induce CD4 internalization, suppresses the accelerated endocytosis of a chimeric integral membrane protein harboring Nef as its cytoplasmic domain and confers a dominant-negative phenotype to the viral protein. Taken together, these data identify mu adaptins as downstream mediators of the down-modulation of CD4, and possibly MHC I, by Nef.     

Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis

The ATP-dependent dissociation of clathrin from clathrin-coated vesicles (CCVs) by the molecular chaperone Hsc70 requires J-domain cofactor proteins, either auxilin or cyclin-G-associated kinase (GAK). Both the nerve-specific auxilin and the ubiquitous GAK induce CCVs to bind to Hsc70. The removal of auxilin or GAK from various organisms and cells has provided definitive evidence that Hsc70 uncoats CCVs in vivo. In addition, evidence from various studies has suggested that Hsc70 and auxilin are involved in several other key processes that occur during clathrin-mediated endocytosis. First, Hsc70 and auxilin are required for the clathrin exchange that occurs during coated-pit invagination and constriction; this clathrin exchange may catalyze any rearrangement of the clathrin-coated pit (CCP) structure that is required during invagination and constriction. Second, Hsc70 and auxilin may chaperone clathrin after it dissociates from CCPs so that it does not aggregate in the cytosol. Third, auxilin and Hsc70 may be involved in the rebinding of clathrin to the plasma membrane to form new CCPs and independently appear to chaperone adaptor proteins so that they can also rebind to membranes to nucleate the formation of new CCPs. Finally, if formation of the curved clathrin coat induces membrane curvature, then Hsc70 and auxilin provide the energy for this curvature by inducing ATP-dependent clathrin exchange and rearrangement during endocytosis and ATP-dependent dissociation of clathrin at the end of the cycle so that it is energetically primed to rebind to the plasma membrane.     

Nef-mediated Clathrin-coated Pit Formation

The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.     

Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation.

The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.     

Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size

Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP(2) synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5'-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP(2) synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.     

CD4 down-regulation by HIV-1 and simian immunodeficiency virus (SIV) Nef proteins involves both internalization and intracellular retention mechanisms

Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.     

Clathrin‐Coated Pits: Vive La Différence?

Because of the discovery of coated pits and vesicles more than 40 years ago and the identification of clathrin as a major component of the coat, it has been assumed that clathrin-coated pits (CCPs) are responsible for the uptake of most plasma membrane receptors undergoing internalization. The recent molecular characterization of clathrin-independent routes of endocytosis confirms that several alternative endocytic pathways operate at the plasma membrane of mammalian cells. This heterogeneous view of endocytosis has been expanded still further by recent studies, suggesting that different subpopulations of CCPs responsible for the internalization of specific sets of cargo may coexist. In the present review, we have discussed the experimental evidence in favor or against the existence of distinct parallel clathrin-dependent pathways at the plasma membrane.     

Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef is a myristylated protein with a relative molecular mass of 27 kDa, is localized to the cytoplasmic surfaces of cellular membranes, and has been reported to down-modulate CD4 in human T cells. To understand the mechanism of HIV-1 Nef-mediated down-modulation of cell surface CD4, we expressed Nef protein in human T-cell line VB. Expression of HIV-1 Nef protein down-modulated surface CD4 molecules. In pulse-chase experiments, CD4 molecules in Nef-expressing cells were synthesized at normal levels. However, the bulk of newly synthesized CD4 protein was degraded with a half-life of approximately 6 h, compared with the 24-h half-life in control cells. This Nef-induced acceleration of CD4 turnover was inhibited by lysosomotropic agents NH4Cl and chloroquine as well as by the protease inhibitor leupeptin. Surface CD4 biotinylation experiments demonstrated that CD4 molecules in Nef-expressing T cells are transported to the plasma membrane with normal kinetics but are then rapidly internalized. Therefore, HIV-1 Nef-induced down-modulation of CD4 is due to rapid internalization of surface CD4 and subsequent degradation by an acid-dependent process, potentially lysosomal. Additionally, in a Nef-expressing cell, we find accelerated dissociation of the T-cell tyrosine kinase p56lck and CD4 but only after the complex reaches the plasma membrane. This implies that HIV-1 Nef protein might play a role in triggering a series of T-cell activation-like events, which contribute to p56lck dissociation and internalization of surface CD4 molecules.     

Understanding living clathrin-coated pits

Most knowledge of clathrin-mediated endocytosis has been gained by biochemical fractionation and in vitro assays. Recently, the study of endocytosis has extended into the living cell. The tracking of individual clathrin-coated pits and vesicles (CCPs and CCVs) has provided new insight into understanding the dynamic nature of CCPs. The use of total internal reflection fluorescence microscopy (TIR-FM), also termed evanescent field microscopy, has enabled the direct observation of events occurring within a restricted area of the cell adjacent to and including the adherent plasma membrane. TIR-FM is now actively being pursued in the study of endocytic processes. The direct observation of CCP-associated proteins including clathrin itself, dynamin and, most recently, AP-2 has considerably challenged old models, confirming some points but raising very interesting new questions.     

Cutting edge: SIV Nef protein utilizes both leucine- and tyrosine-based protein sorting pathways for down-regulation of CD4.

The Nef protein is unique to primate lentiviruses and is closely linked to accelerated pathogenesis in both human and monkey hosts. Nef acts to down-regulate CD4 and MHC class I, two receptors important for immune function. A recent report demonstrated the presence of two tyrosine motifs in SIV Nef that contribute to its ability to down-regulate CD4 and to associate with clathrin adaptors. These tyrosine motifs are not present in HIV-1 Nef, which instead utilizes a leucine-based motif for its down-regulation of CD4. We now report that SIV Nef also contains a conserved leucine-based motif that contributes to CD4 down-regulation, functions to stimulate internalization, and contributes to the association of SIV Nef with clathrin adaptors AP-1 and AP-2. These results demonstrate that SIV Nef differs from HIV-1 Nef by its ability to use two parallel pathways of the protein-sorting machinery based on either tyrosine or leucine motifs.     

Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum

HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb₃, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, βCOP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed .     

A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3γ, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA–DM and the invariant chain (I chain) complexed to HLA–DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR–I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR–I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR–I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR–I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR–I chain until their intended rendezvous in late endocytic compartments.     

Human immunodeficiency virus type 1 nef expression prevents AP-2-mediated internalization of the major histocompatibility complex class II-associated invariant chain

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.     

Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy

Using a new scheme based on atomic force microscopy (AFM), we investigate mechanical properties of clathrin-coated vesicles (CCVs). CCVs are multicomponent protein and lipid complexes of approximately 100 nm diameter that are implicated in many essential cell-trafficking processes. Our AFM imaging resolves clathrin lattice polygons and provides height deformation in quantitative response to AFM-substrate compression force. We model CCVs as multilayered elastic spherical shells and, from AFM measurements, estimate their bending rigidity to be 285 +/- 30 k(B)T, i.e., approximately 20 times that of either the outer clathrin cage or inner vesicle membrane. Further analysis reveals a flexible coupling between the clathrin coat and the membrane, a structural property whose modulation may affect vesicle biogenesis and cellular function.     

Inhibition of clathrin‐mediated endocytosis by knockdown of AP‐2 leads to alterations in the plasma membrane proteome

In eukaryotic cells, clathrin‐mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin‐coated vesicles (CCVs) is AP‐2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP‐2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal‐lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti‐cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP‐2 knockdown attenuated the global endocytic capacity, but clathrin‐independent entry pathways were still operating, as indicated by persistent internalization of specific membrane‐spanning and GPI‐anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP‐2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.     

Auxilin depletion causes self-assembly of clathrin into membraneless cages in vivo

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.     

Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells

During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.     

Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef use distinct but overlapping target sites for downregulation of cell surface CD4.

Although the Nef proteins encoded by human immunodeficiency virus type 1 (HIV-1) and simian immuno-deficiency virus (SIV) are known to induce the efficient internalization and degradation of cell surface CD4, it remains unclear whether this process involves a direct interaction between Nef and CD4. Here, we report that CD4 downregulation by HIV-1 and SIV Nef requires distinct but overlapping target sites within the CD4 intracytoplasmic domain. In particular, mutation of a glutamic acid residue located at CD4 residue 405 or of arginine and methionine residues located, respectively, at residue 406 and 407 results in a mutant CD4 protein that is efficiently downregulated by HIV-1 Nef but refractory to downregulation by SIV Nef. However, both HIV-1 and SIV Nef require an isoleucine located at residue 410 and the dileucine motif found at CD4 residues 413 and 414. CD4 downregulation induced by the Nef protein encoded by HIV-2 is shown to require a CD4 target sequence that is similar to, but distinct from, that observed with SIV Nef. These data explain the previous finding that the murine CD4 protein, which has an alanine at residue 405, is refractory to downregulation by SIV, but not HIV-1, Nef (J. L. Foster, S.J. Anderson, A. L. B. Frazier, and J. V. Garcia, Virology 201:373-379, 1994). In addition, these observations provide strong genetic support for the hypothesis that the Nef-mediated downregulation of cell surface CD4 requires a direct Nef-CD4 interaction.     

Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.