The ectodomain of the human T-cell leukemia virus type 1 TM glycoprotein is involved in postfusion events.

To examine the contribution of the transmembrane envelope glycoprotein (TM) to the infectivity of the human T-cell leukemia virus type 1 (HTLV-1), single amino acid substitutions were introduced throughout its ectodomain. The mutated envelopes were tested for intracellular maturation and for functions, including ability to elicit syncytium formation and ability to mediate cell-to-cell transmission of the virus. Three major phenotypes, defining three functionally distinct regions, were identified. (i) Mutations causing defects in intracellular maturation of the envelope precursor are mostly distributed in the central portion of the TM ectodomain, containing the immunosuppressive peptide. This region, which includes vicinal cysteines thought to form an intramolecular disulfide bridge, is probably essential for correct folding of the protein. (ii) Mutations resulting in reduced syncytium-forming ability despite correct intracellular maturation are clustered in the amino-terminal part of the TM ectodomain, within the leucine zipper-like motif. Similar motifs with a propensity to form coiled-coil structures have been implicated in the fusion process driven by other viral envelope proteins, and HTLV-1 may thus conform to this general rule for viral fusion. (iii) Mutants with increased syncytium-forming ability define a region immediately amino-terminal to the membrane-spanning domain. Surprisingly, these mutants exhibited severe defects in infectivity, despite competence for fusion. Existence of this phenotype indicates that capacity for cell-to-cell fusion is not sufficient to ensure viral entry, even in cell-to-cell transmission. The ectodomain of the TM glycoprotein thus may be involved in postfusion events required for full infectivity of HTLV-1, which perhaps represents a unique feature of this poorly infectious retrovirus.     

Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11

A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.     

Differential effects of N-glycans on surface expression suggest structural differences between the acid-sensing ion channel (ASIC) 1a and ASIC1b

ASICs (acid-sensing ion channels) are H(+)-gated Na(+) channels with a widespread expression pattern in the central and the peripheral nervous system. ASICs have a simple topology with two transmembrane domains, cytoplasmic termini and a large ectodomain between the transmembrane domains; this topology has been confirmed by the crystal structure of chicken ASIC1. ASIC1a and ASIC1b are two variants encoded by the asic1 gene. The variable part of the protein includes the cytoplasmic N-terminus, the first transmembrane domain and approximately the first third of the ectodomain. Both variants contain two consensus sequences for N-linked glycosylation in the common, distal part of the ectodomain. In contrast with ASIC1a, ASIC1b contains two additional consensus sequences in the variable, proximal part of the ectodomain. Here we show that all the extracellular asparagine residues within the putative consensus sequences for N-glycosylation carry glycans. The two common distal glycans increase surface expression of the channels, but are no absolute requirement for channel activity. In sharp contrast, the presence of at least one of the two proximal glycans, which are specific to ASIC1b, is an absolute requirement for surface expression of ASIC1b. This result suggests substantial differences in the structure of the proximal ectodomain between the two ASIC1 variants.     

Screening for mutants defective in secretory protein maturation and ER quality control

A genetic strategy devised to understand the physiology of the unfolded protein response serendipitously generated mutants affecting a broad spectrum of functions needed for secretory protein biogenesis and quality control. These included N- and O-linked glycosylation, glycosylphosphatidylinositol anchor biosynthesis and transfer, protein folding, protein trafficking, lumenal ionic homeostasis, ER quality control, and ER associated protein degradation. As these pathways are incompletely understood, the screen provides a simple method for their genetic dissection. This article describes methods for isolating novel mutants of these pathways and strategies for identifying corresponding genes.     

Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit

We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.     

Analysis of the membrane topology of the acid-sensing ion channel 2a

Acid-sensing ion channels, or ASICs, are members of the amiloride-sensitive cationic channel superfamily that are predicted to have intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop. This prediction comes from biochemical studies of the mammalian epithelial sodium channels where glycosylation mutants identified the extracellular regions of the channel and a combination of antibody sensitivity and protease action substantiated the intracellular nature of the amino and carboxyl termini. However, although there are highly conserved regions within the different cation channel family members, membrane topology prediction programs provide several alternative structures for the ASICs. Thus, we used glycosylation studies to define the actual membrane topology of the ASIC2a subtype. We deleted the five predicted endogenous asparagine-linked glycosylation sites (Asn-Xaa-(Ser/Thr)) at Asn-22, Asn-365, Asn-392, Asn-478, and Asn-487 to map the extracellular topology. We then introduced exogenous asparagine-linked glycosylation sites at Lys-4, Pro-37, Arg-63, Tyr-67, His-72, Ala-81, Tyr-414, Tyr-423, and Tyr-453 to define the transmembrane domain borders. Finally, we used cell permeabilization studies to confirm the intracellular amino termini of ASIC2a. The data show that Asn-365 and Asn-392 are extracellular and that the introduction of asparagine-linked glycosylation sites at His-72, Ala-81, Tyr-414, and Tyr-423 leads to an increase in molecular mass consistent with an extracellular apposition. In addition, heterologous expression of ASIC2a requires membrane permeabilization for antibody staining. These data confirm the membrane topology prediction that the ASIC2a subtype consists of intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop.     

Molecular regulation of lysophosphatidic acid receptor 1 trafficking to the cell surface

The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell proliferation, migration, and cytokine release. Here, we investigate the molecular signature of LPA1 trafficking to the cell surface. The overexpressed LPA1 with a C-terminal V5 tag (LPA1-V5) is majorly expressed on the cell surface, while two deletion mutants (C320 and ∆84–87) failed to be trafficked to the cell surface. Further, site-directed mutagenesis analysis of the LPA1 revealed that Ile325, Tyr85, and Leu87 within these two fragments regulate LPA1 maturation and trafficking to the cell surface. Over-expression of Sar1, a component of coat protein complex II (COPII), enhances glycosylation of LPA1 wild type, but not these mutants. The mutants of LPA1 are majorly localized in the endoplasmic reticulum (ER) and exhibit a higher binding affinity to heat shock protein 70 (Hsp70), when compared to the LPA1 wild type. Further, we found that all these mutants failed to increase phosphorylation of Erk, and the cytokine release in response to LPA treatment. These results suggest that Ile325, Tyr85, and Leu87 within LPA1 are essential for LPA1 protein properly folding in the ER.     

Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein

The vesicular stomatitis virus glycoprotein (G protein) is an integral membrane protein which assembles into noncovalently associated trimers before transport from the endoplasmic reticulum. In this study we have examined the folding and oligomeric assembly of twelve mutant G proteins with alterations in the cytoplasmic, transmembrane, or ectodomains. Through the use of conformation-specific antibodies, we found that newly synthesized G protein folded into a conformation similar to the mature form within 1-3 min of synthesis and before trimer formation. Mutant proteins not capable of undergoing correct initial folding did not trimerize, were not transported, and were found in large aggregates. They had, as a rule, mutations in the ectodomain, including several with altered glycosylation patterns. In contrast, mutations in the cytoplasmic domain generally had little effect on folding and trimerization. These mutant proteins, whose ectodomains were identical to the wild-type by several assays, were either transported to the cell surface slowly or not at all. We concluded that while correct ectodomain folding and trimer formation are prerequisites for transport, they alone are not sufficient. The results suggest that the cytoplasmic domain of the wild-type protein may facilitate rapid, efficient transport from the ER, which can be easily affected or eliminated by tail mutations that do not detectably affect the ectodomain.     

Alternative splicing and interaction with di- and polyvalent cations control the dynamic range of acid-sensing ion channel 1 (ASIC1)

Homomeric acid-sensing ion channel 1 (ASIC1) can be activated by extracellular H(+) in the physiological pH range and may, therefore, contribute to neurotransmission and peripheral pain perception. ASIC1a and ASIC1b are alternative splice products of the ASIC1 gene. Here we show that both splice variants show steady-state inactivation when exposed to slightly decreased pH, limiting their operational range. Compared with ASIC1a, steady-state inactivation and pH activation of ASIC1b are shifted to more acidic values by 0.25 and 0.7 pH units, respectively, extending the dynamic range of ASIC1. Shifts of inactivation and activation are intimately linked; only two amino acids in the ectodomain, which are exchanged by alternative splicing, control both properties. Moreover, we show that extracellular, divalent cations like Ca(2+) and Mg(2+) as well as the polyvalent cation spermine shift the steady-state inactivation of ASIC1a and ASIC1b to more acidic values. This leads to a potentiation of the channel response and is due to a stabilization of the resting state. Our results indicate that ASIC1b is an effective sensor of transient H(+) signals during slight acidosis and that, in addition to alternative splicing, interaction with di- and polyvalent cations extends the dynamic range of ASIC H(+) sensors.     

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.     

Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.     

Assembly, target-signaling and intracellular transport of tyrosinase gene family proteins in the initial stage of melanosome biogenesis

Assembly, target-signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein-3 (AP-3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di-leucine motif in their cytoplasmic tail, to which AP-3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Functional Domains in the Retroviral Transmembrane Protein

The envelope glycoproteins of the mammalian type C retroviruses consist of two subunits, a surface (SU) protein and a transmembrane (TM) protein. SU binds to the viral receptor and is thought to trigger conformational changes in the associated TM protein that ultimately lead to the fusion of viral and host cell membranes. For Moloney murine leukemia virus (MoMuLV), the envelope protein probably exists as a trimer. We have previously demonstrated that the coexpression of envelope proteins that are individually defective in either the SU or TM subunits can lead to functional complementation (Y. Zhao et al., J. Virol. 71:6967–6972, 1997). We have now extended these studies to investigate the abilities of a panel of fusion-defective TM mutants to complement each other. This analysis identified distinct complementation groups within TM, with implications for interactions between different regions of TM in the fusion process. In viral particles, the C-terminal 16 amino acids of the MoMuLV TM (the R peptide) are cleaved by the viral protease, resulting in an increased fusogenicity of the envelope protein. We have examined the consequences of R peptide cleavage for the different TM fusion mutants and have found that this enhancement of fusogenicity can only occur in cis to certain of the TM mutants. These results suggest that R peptide cleavage enhances the fusogenicity of the envelope protein by influencing the interaction of two distinct regions in the TM ectodomain.     

Intragenic Suppressing Mutations Correct the Folding and Intracellular Traffic of Misfolded Mutants of Yor1p,a Eukaryotic Drug Transporter

ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and defective assembly of these proteins can cause severe disease associated with improper drug or ion flux. The yeast protein Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention in the endoplasmic reticulum (ER) of the resulting protein Yor1p-ΔF670, similar to the predominant disease-causing allele in humans, CFTR-ΔF508. Here we describe two novel Yor1p mutants, G278R and I1084P, which fail to assemble and traffic similar to Yor1p-ΔF670. These mutations are located in the two intracellular loops (ICLs) that interface directly with NBD1, and thus disrupt a functionally important structural module. We isolated 2 second-site mutations, F270S and R1168M, which partially correct the folding injuries associated with the G278R, I1084P, and ΔF670 mutants and reinstate their trafficking. The position of both corrective mutations at the cytoplasmic face of a transmembrane helix suggests that they restore biogenesis by influencing the behavior of the transmembrane domains rather than by direct restoration of the ICL1-ICL4-NBD1 structural module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally important inter-domain interactions and suggest new potential avenues for correcting folding defects caused by abrogation of those domain interfaces.     

Receptor-associated protein facilitates proper folding and maturation of the low-density lipoprotein receptor and its class 2 mutants

Familial hypercholesterolemia is the consequence of various mutations in the low-density lipoprotein receptor (LDLR). In the current study, we show that a specialized molecular chaperone, the receptor-associated protein (RAP), promotes proper folding and subsequent exocytic trafficking of the wild-type LDLR and several of its class 2 mutants. Co-immunoprecipitation with anti-RAP antibody demonstrates that RAP interacts with the LDLR. Kinetic analyses of LDLR posttranslational folding and maturation in the absence or presence of RAP coexpression show that RAP prevents aggregation and promotes the maturation of the LDLR. Additionally, depletion of Ca(2+) in intact cells impairs LDLR folding, and coexpression of RAP partially corrects this misfolding. Finally, we show that the increased mature cell surface LDLR in the presence of RAP coexpression is functional in its ability to endocytose and degrade (125)I-LDL. Taken together, our results show that the folding, trafficking, and maturation of the LDLR and its class 2 mutants are promoted by RAP.     

Physical and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis

Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored. Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control. Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells. We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps. Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes. Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle. These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits.     

Structural Elements for the Generation of Sustained Currents by the Acid Pain Sensor ASIC3

ASIC3 is an acid-sensing ion channel expressed in sensory neurons, where it participates in acidic and inflammatory pain. In addition to the “classical” transient current, ASIC3 generates a sustained current essential for pain perception. Using chimeras between the ASIC3 and ASIC1a channels we show that the first transmembrane domain (TM1), combined with the N-terminal domain, is the key structural element generating the low pH (<6.5)-evoked sustained current. The TM1 domain also modulates the pH-dependent activation of the fast transient current thus contributing to a constitutive window current, another type of sustained current present near physiological pH. The C-terminal and the TM2 domains negatively regulate both types of sustained current, and the extracellular loop affects its kinetics. These data provide new information to aid understanding the mechanisms of the multifaceted pH gating of ASIC3. Together with the peak current, both components of the sustained current (window and sustained at pH <6.5) allow ASIC3 to adapt its behavior to a wide range of extracellular pH variations by generating transient and/or sustained responses that contribute to nociceptor excitability.     

Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8

ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.     

N-linked glycan-dependent interaction of CD63 with CXCR4 at the Golgi apparatus induces downregulation of CXCR4

Efficient downregulation of CXCR4 cell surface expression by introduction of the CD63 gene has previously been reported by us. In the present study, it was found that CD63 and its mutant efficiently interact with CXCR4 in live cells and that CD63-induced downregulation and interaction are significantly abrogated by the N- linked glycosylation inhibitor, TM. Furthermore, the downregulation and interaction were clearly attenuated by alternation of all three N- linked glycosylation sites in CD63. Either CD63 or CD63ΔN formed a complex with CXCR4 at the Golgi apparatus and the late endosomes, while CD63 GD mutants lost the ability to form a complex with CXCR4 exclusively at the Golgi apparatus. These findings suggest that CD63 interacts with CXCR4 through the N- linked glycans-portion of the CD63 protein and that the complex induces direction of CXCR4 trafficking to the endosomes/lysosomes, rather than to the plasma membrane. At the Golgi apparatus, there may be lysosome protein (CD63)-associated machinery that influences trafficking of other membrane proteins.