ABSTRACTHerpes simplex virus (HSV) and varicella-zoster virus (VZV) are two pathogenic human alphaherpesviruses whose intracellular assembly is thought to follow different pathways. VZV presumably acquires its envelope in thetrans-Golgi network (TGN), and it has recently been shown that its major envelope glycoprotein, VZV-gE, accumulates in this compartment when expressed alone. In contrast, the envelopment of HSV has been proposed to occur at the inner nuclear membrane, although to which compartment the gE homolog (HSV-gE) is transported is unknown. For this reason, we have studied the intracellular traffic of HSV-gE and have found that this glycoprotein accumulates at steady state in the TGN, both when expressed from cloned cDNA and in HSV-infected cells. In addition, HSV-gE cycles between the TGN and the cell surface and requires a conserved tyrosine-containing motif within its cytoplasmic tail for proper trafficking. These results show that VZV-gE and HSV-gE have similar intracellular trafficking pathways, probably reflecting the presence of similar sorting signals in the cytoplasmic domains of both molecules, and suggest that the respective viruses, VZV and HSV, could use the same subcellular organelle, the TGN, for their envelopment.

In mammalian cells, the mannose 6-phosphate receptors (MPRs) and the lysosomal glycoproteins, lysosomal-associated membrane protein (LAMP) I, lysosomal integral membrane protein (LIMP) II, are directly transported from the trans-Golgi network to endosomes and lysosomes. While MPR traffic relies on the AP-1 adaptor complex, we report that proper targeting of LAMP I and LIMP II to lysosomes requires the AP-3 adaptor-like complex. Overexpression of these proteins, which contain either a tyrosine- or a di-leucine-based-sorting motif, promotes AP-3 recruitment on membranes. Inhibition of AP-3 function using antisense oligonucleotides leads to a selective misrouting of both LAMP I and LIMP II to the cell surface without affecting MPR trafficking. These results provide evidence that AP-3 functions in the intracellular targeting of transmembrane glycoproteins to lysosomes.

The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses.

The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses. The varicella-zoster virus (VZV) is the etiological agent of two different human pathologies, chickenpox (varicella) and shingles (zoster). This alphaherpesvirus is believed to acquire its lipidic envelope in the trans-Golgi network (TGN). This is consistent with previous data showing that the most abundant VZV envelope glycoprotein gE accumulates at steady-state in this organelle when expressed from cloned cDNA. In the present study, we have investigated the intracellular trafficking of gI, another VZV envelope glycoprotein. In transfected cells, this protein shows a very slow biosynthetic transport to the cell surface where it accumulates. However, upon co-expression of gE, gI experiences a dramatic increase in its exit rate from the endoplasmic reticulum, it accumulates in a sialyltransferase-positive compartment, presumably the TGN, and cycles between this compartment and the cell surface. This differential behavior results from the ability of gE and gI to form a complex in the early stages of the biosynthetic pathway whose intracellular traffic is exclusively determined by the sorting information in the tail of gE. Thus, gI provides the first example of a molecule localized to the TGN by means of its association with another TGN protein. We also show that, during the early stages of VZV infection, both proteins are also found in the TGN of the host cell. This suggests the existence of an intermediate stage during VZV biogenesis in which the envelope glycoproteins, transiently arrested in the TGN, could promote the envelopment of newly synthesized nucleocapsids into this compartment and, therefore, the assembly of infective viruses. The trans-Golgi network (TGN) 1The abbreviations used are: TGN, trans-Golgi network; BFA, brefeldin A; BHV, bovine herpesvirus; ER, endoplasmic reticulum; FHV, feline herpesvirus; HSV, herpes simplex virus; PRV, pseudorabies virus; ST, sialyltransferase; VZV, varicella-zoster virus; FITC, fluorescein isothiocyanate; TRITC, tetrahodamine isothiocyanate; MEM, minimum Eagle's medium; VSV-G, vesicular stomatitis virus glycoprotein G. is a tubuloreticular compartment located on the trans-most side of the Golgi complex (1Rambourg A. Clermont Y. Eur. J. Cell Biol. 1990; 51: 189-200PubMed Google Scholar). This organelle houses different proteins that are involved in adding post-translational modifications to polypeptides traveling along the secretory pathway. In addition, this organelle constitutes the main sorting station in the secretory pathway (2Mellman I. Simons K. Cell. 1992; 68: 829-840Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 3Griffiths G. Simons K. Science. 1986; 234: 438-443Crossref PubMed Scopus (764) Google Scholar). The TGN can undergo rapid tubularization and mixing with endosomal compartments in the presence of the fungal metabolite brefeldin A (BFA) (4Klausner R.D. Donaldson J.G. Lippincott-Scwharz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1542) Google Scholar). The TGN has also been used by certain viruses as a membrane donor for their lipidic envelope (envelopment or budding process), as it happens in the case of the varicella-zoster virus (VZV) (5Gershon A.A. Sherman D.L. Zhu Z. Gabel C.A. Ambron R.T. Gershon M.D. J. Virol. 1994; 68: 6372-6390Crossref PubMed Google Scholar, 6Zhu Z. Gershon M.D. Gabel C. Sherman D. Ambron R. Gershin A. Neurology. 1995; 45: S15-S17Crossref PubMed Google Scholar). VZV is a human alphaherpesvirus causing chickenpox (varicella), as a result of the primary infection, and shingles (zoster) upon reactivation of the latent virus (7Weller T.H. N. Engl. J. Med. 1983; 309: 1362-1368Crossref PubMed Scopus (219) Google Scholar, 8Weller T.H. N. Engl. J. Med. 1983; 309: 1434-1440Crossref PubMed Scopus (173) Google Scholar). As it occurs in other alphaherpesviruses, the VZV nucleocapsids are assembled in the nuclei of the infected cells. These nucleocapsids are then released into the periplasmic space by budding through the inner nuclear membrane, thereby acquiring a transient envelope that is lost upon fusion with the outer nuclear membrane. In this way, the nucleocapsids are released in the cytosol, where they acquire a second and definitive envelope. This envelope is derived from the TGN, as initially demonstrated by Gershon et al. (5Gershon A.A. Sherman D.L. Zhu Z. Gabel C.A. Ambron R.T. Gershon M.D. J. Virol. 1994; 68: 6372-6390Crossref PubMed Google Scholar) by examining VZV-infected cells at the ultrastructural level. Mature viruses accumulate finally in an intracellular endosomal compartment (9Gabel C.A. Dubey L. Steinberg S.P. Sherman D. Gershon M.D. Gershon A.A. J. Virol. 1989; 63: 4264-4276Crossref PubMed Google Scholar). As happens in other cases of viruses that undergo intracellular assembly, envelopment of VZV in the TGN requires that the corresponding envelope glycoproteins have to be delivered to this compartment during viral infection (10Zhu Z. Gershon M.D. Hao Y. Ambron R.T. Gabel C.A. Gershon A.A. J. Virol. 1995; 69: 7951-7959Crossref PubMed Google Scholar, 11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). This implies that sorting signals must exist within these glycoproteins to ensure their correct targeting, making these molecules very useful tools for analyzing the mechanisms involved in TGN localization. We and others have recently shown that the most abundant envelope glycoprotein of VZV (gpI or gE) accumulates in the TGN when expressed from cloned cDNA and that this accumulation results, at least partially, from its ability to be rapidly retrieved form the cell surface (10Zhu Z. Gershon M.D. Hao Y. Ambron R.T. Gabel C.A. Gershon A.A. J. Virol. 1995; 69: 7951-7959Crossref PubMed Google Scholar, 11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). The sorting information in the sequence of gE has been mapped to its cytoplasmic tail, and shown to consist of two tyrosine-containing tetrapeptides related to endocytosis motifs (11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar) and a more C-terminal acidic cluster that contains casein-kinase II- phosphorylatable residues (11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). These signals are similar to those found in other molecules known to be localized in the TGN at steady state, such as TGN38 or furin (13Jones B.G. Thomas L. Molloy S.S. Thulin C.D. Fry M.D. Walsh K.A. Thomas G. EMBO J. 1995; 14: 5869-5883Crossref PubMed Scopus (164) Google Scholar, 14Schäfer W. Stroh A. Bergho¨fer S. Seiler J. Vey M. Kruse M.-L. Kern H.F. Klenk H.-D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (220) Google Scholar, 15Takahashi S. Nakagawa T. Banno T. Watanabe T. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 28397-28401Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 16Voorhoes P. Deignan E. van Donselaar E. Humphrey J. Marks M. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar, 17Ponnambalam S. Rabouille C. Luzio J.P. Nilsson T. Warren G. J. Cell Biol. 1994; 125: 253-268Crossref PubMed Scopus (120) Google Scholar, 18Bos K. Wraight C. Stanley K. EMBO J. 1993; 12: 2219-2228Crossref PubMed Scopus (193) Google Scholar, 19Wong S.H. Hong W. J. Biol. Chem. 1993; 268: 22853-22862Abstract Full Text PDF PubMed Google Scholar). In addition to gE, there are at least five additional glycoproteins in the envelope of VZV (gB, gH, gI, gC, and gL, formerly known as gpII, gpIII, gpIV, gpV, and gpVI, respectively) (20Davidson A.J. Edson C.M. Ellis R.W. Forghani B. Gilden D. Grose C. Keller P.M. Vafai A. Wroblewska Z. Yamanishi K. J. Virol. 1986; 57: 1195-1197Crossref PubMed Google Scholar), whose sequences are apparently devoid of TGN-sorting information. If VZV indeed acquires its final envelope in the TGN, then mechanisms must exist to ensure that all these molecules reach this compartment in order to promote infective VZV formation. In the present article, we have focused our attention on another type I glycoprotein of the viral envelope, the glycoprotein gI (or gpIV). This molecule has been shown to physically interact with gE in VZV (21Yao Z. Jackson W. Forghani B. Grose C. J. Virol. 1993; 67: 305-314Crossref PubMed Google Scholar), as well as in herpes simplex virus (HSV-1) (22Johnson D.C. Feenstra V. J. Virol. 1987; 61: 2208-2216Crossref PubMed Google Scholar), feline herpesvirus (FHV-1) (23De Mijnes J.D.F. van der Horst L.M. van Anken E. Horzinek M.C. Rottier P.J.M. de Groot R.J. J. Virol. 1996; 70: 5466-5475Crossref PubMed Google Scholar), and pseudorabies virus (PRV) (24Whealy M.E. Card J.P. Robbins A.K. Dubin J.R. Rziha H.-J. Enquist L.W. J. Virol. 1993; 67: 3786-3797Crossref PubMed Google Scholar), three other members of the alphaherpesvirinae subfamily. Our results indicate that gI, which is found in the cell surface when expressed alone, accumulates in the TGN when expressed together with gE. This accumulation of gI in the TGN also relies on its rapid internalization from the cell surface. Our data indicate that gE and gI precursors can form a stoichiometric complex in the endoplasmic reticulum (ER), which results in an increased maturation rate of gI. We have also found that, in VZV-infected cells, both gE and gI can be found shortly after infection in a perinuclear compartment that most likely corresponds to the TGN. Monoclonal antibodies SG1 and SG4, against VZV gE and gI, respectively, were obtained from Viro Research Inc. (Rockford, IL). The polyclonal serum against the cytoplasmic tail of furin was generously provided by Dr. W. Garten (University of Marburg, Marburg, Germany). The SA48 HeLa clone stably expressing VSVG-tagged sialyltransferase (ST-VSVG) was a generous gift of Dr. Tommy Nilsson (EMBL, Heidelberg, Germany). All secondary antibodies against the Fc of mouse or rabbit IgGs coupled to FITC or rhodamine were purchased from Dianova (Hamburg, Germany). Construction of the gE expression vector has been previously described (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). In order to clone VZV gI, the complete open reading frame was amplified from a lysate of VZV (Dumas strain)-infected cells using the Expand High Fidelity kit (Boehringer Mannheim, Mannheim, Germany). The resulting fragment was digested withXbaI and HindIII and was cloned into the same sites of the eukaryotic expression vector pSFFV6 (25Chen H.J. Remmler J. Delaney J.C. Messner D.J. Lobel P. J. Biol. Chem. 1993; 268: 22338-22346Abstract Full Text PDF PubMed Google Scholar), or downstream the T7 promoter in pGEM1. The gE mutants containing the cytoplasmic tail of the yeast protein Wbp1p with either the C-terminal KKXX or the SSXX signals were constructed by polymerase chain reaction-based amplification using reverse primers in which the corresponding sequences of the wild-type or mutated Wbp1p cytoplasmic tails had been introduced as translational fusions with the sequence of the gE transmembrane domain. The resulting polymerase chain reaction fragments were digested with XbaI and HindIII and cloned into the same sites in the pSFFV6 vector. The sequences of both mutants were verified using the Sanger dideoxy chain termination method. The antibody 1667 against the full-length gE was obtained by cloning a cDNA fragment coding for the mature VZV gE open reading frame with a hexahistidine tag at the C terminus into the NcoI/BamHI sites of the pET15b vector (Novagen, Wiesbaden, Germany). The protein was expressed in BL21 cells and the insoluble fraction (containing most of the recombinant gE) was solubilized in 8 m urea and loaded on a Talon metal-affinity column (CLONTECH, Heidelberg, Germany). After extensive washing, the bound protein was eluted with SDS-loading buffer, and approximately 50 μg were loaded on a 7.5% preparative SDS-polyacrylamide gel. The part of the gel containing the recombinant protein was excised, homogenized using a Teflon-glass homogenizer, mixed with either Freund's complete or incomplete adjuvant, and used to immunize rabbits following standard procedures. To produce the 2679 antibody against the cytoplasmic tail of gI, a fragment comprising amino acids 314–354 of the gI precursor form was cloned into the pGEX-4T-1 vector (Pharmacia, Freiburg, Germany) as a fusion to glutathione S-transferase. The glutathioneS-transferase-gI fusion was expressed in XL-1 Blue cells and purified by affinity chromatography on a glutathione-Sepharose column (Pharmacia, Freiburg, Germany), following the manufacturer's instructions. After elution, the fusion protein was loaded on a preparative 7.5% preparative SDS-polyacrylamide gel. The gel fragment containing the band was excised, homogenized, mixed with Freund's adjuvant, and used to inoculate rabbits following a standard immunization schedule. The serum was affinity-purified by incubation with a nitrocellulose strip onto which the recombinant glutathioneS-transferase-gI had been previously bound (26Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). For the internalization assays, a continuous uptake was performed in which transfected cells seeded on coverslips were washed with prewarmed α-MEM and overlaid with 200 μl of complete α-MEM in which the antibodies had been diluted as specified in the figure legends. After 1-h incubation, the internalization medium was removed and the cells were immediately fixed and processed for immunofluorescence using fluorescein or rhodamine-coupled secondary antibodies. Due to the cell-associated nature of VZV, infections were carried out as described previously (27Defechereux P. Debrus S. Baudoux L. Schoonbroodt S. Merville M.-P. Rentier B. Piette J. J. Gen. Virol. 1996; 77: 1505-1513Crossref PubMed Scopus (14) Google Scholar), by co-culture of VZV (Ellen strain)-infected Vero cells with noninfected either Vero or HeLa cells. For the immunofluorescence experiments, infected and noninfected cells were plated on coverslips at a 1:4 ratio and grown in complete α-MEM for different times as indicated in the figure legends. The cells were subsequently fixed and processed for immunofluorescence. Published procedures were used for vaccinia T7 infection (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), metabolic labeling of the cells and immunoprecipitation (28Mauxion F. Le Borgne R. Munier-Lehmann H. Hoflack B. J. Biol. Chem. 1996; 271: 2171-2178Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and for calcium-phosphate transient transfection and indirect immunofluorescence (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). To address the subcellular localization of the VZV-envelope glycoprotein gI (gpIV), we have cloned the complete gI open reading frame in the mammalian expression vector pSFFV6 (25Chen H.J. Remmler J. Delaney J.C. Messner D.J. Lobel P. J. Biol. Chem. 1993; 268: 22338-22346Abstract Full Text PDF PubMed Google Scholar). We have used this construct to perform transient transfection assays in HeLa cells followed by immunofluorescence using anti-gI-specific antibodies. This experiment revealed that, in every transfected cell, gI was exclusively found at the cell surface (Fig.1 b). As a control, we also performed transient transfections with an analogous construct in which the complete gE (gpI) open reading frame had been inserted into the same expression vector (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). In agreement with previous data (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), in cells transfected with the gE expression vector, this protein was exclusively localized in the perinuclear region of the cell (Fig.1 c), in a compartment that has been previously identified as the TGN, based on its co-localization at the light microscopy level with the TGN markers TGN38, furin, and sialyltransferase (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), at the electron microscopic level with galactosyltransferase, 2A. Alconada, S. Ro¨ttgers, and B. Hoflack, unpublished observations. and by its sensitivity to BFA and nocodazole, two drugs that affect the morphology of this compartment (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). We next looked at the localization of gI in HeLa cells that had been simultaneously transfected with gE and gI expression constructs. In these cells, expression of gI was mainly restricted to the perinuclear region of the cell, largely colocalizing with gE, and almost absent from the cell surface (Fig. 1, e and f). We also performed an analogous double-transfection experiment using gI and an unrelated TGN marker (the convertase furin), whose intracellular traffic closely resembles that of gE (13Jones B.G. Thomas L. Molloy S.S. Thulin C.D. Fry M.D. Walsh K.A. Thomas G. EMBO J. 1995; 14: 5869-5883Crossref PubMed Scopus (164) Google Scholar, 14Schäfer W. Stroh A. Bergho¨fer S. Seiler J. Vey M. Kruse M.-L. Kern H.F. Klenk H.-D. Garten W. EMBO J. 1995; 14: 2424-2435Crossref PubMed Scopus (220) Google Scholar, 15Takahashi S. Nakagawa T. Banno T. Watanabe T. Murakami K. Nakayama K. J. Biol. Chem. 1995; 270: 28397-28401Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 16Voorhoes P. Deignan E. van Donselaar E. Humphrey J. Marks M. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar). In this case, whereas expression of furin was restricted to the perinuclear region of the cell (Fig. 1 g), gI was exclusively detected at the cell surface (Fig. 1 h), therefore excluding the possibility that the perinuclear localization of gI in gE-expressing cells was simply due to an inability of the cell to properly sort gI at the TGN in the presence of another highly expressed molecule in this compartment. It has been previously suggested that gE and gI might share common antigenic determinants (29Vafai A. Wroblewska Z. Mahalingan G. Cabirac G. Wellish M. Cisco M. Gilden D. J. Virol. 1988; 62: 2544-2551Crossref PubMed Google Scholar, 30Vafai A. Jensen K. Kubo R. Virus Res. 1989; 13: 319-336Crossref PubMed Scopus (9) Google Scholar), which could explain the perinuclear signal attributed to gI in cells expressing gE if the antibodies used in this study would recognize any of these shared epitopes. However, this does not seem to be the case, since in cells expressing gI, no signal was detected with the anti-gE antibody (Fig. 1 a) and, conversely, no signal was observed with anti-gI antibodies in cells exclusively transfected with gE (Fig. 1 d). To exclude that the strong cell surface gI-staining observed in cells expressing gI alone or gI and furin could mask any labeling of intracellular compartments, we analyzed the single or double-transfected cells by laser scanning confocal microscopy. As expected, no gI-staining could be detected intracellularly (data not shown). The same distribution was observed when polyclonal antibodies against the cytoplasmic domain of gI were used (data not shown). From all these results, we concluded that the localization of gI in transfected cells can be shifted from the cell-surface to the perinuclear region by the simultaneous co-expression of gE. The colocalization experiments shown above indicate that, when expressed together, gE and gI are localized to the same cellular compartment, but they do not prove that this compartment is indeed the TGN, the organelle where gE accumulates when expressed alone (10Zhu Z. Gershon M.D. Hao Y. Ambron R.T. Gabel C.A. Gershon A.A. J. Virol. 1995; 69: 7951-7959Crossref PubMed Google Scholar, 11Zhu Z. Hao Y. Gershon M.D. Ambron R.T. Gershon A.A. J. Virol. 1996; 70: 6563-6575Crossref PubMed Google Scholar, 12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). We have previously used the rapid tubularization in response to BFA as a hallmark of the TGN to distinguish it from other membrane-bound compartments clustered in the perinuclear region of the cell (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar). When HeLa cells expressing gE and gI were treated for 5 min with 10 μg/ml BFA, fixed and decorated with anti-gE and anti-gI antibodies, both molecules were found to colocalize in thin tubules that emanated from the perinuclear region into the cell periphery (Fig.2, a and b), strongly suggesting that the TGN is the compartment where gE and gI accumulate upon co-expression. Another property of certain TGN markers (gE, furin, and TGN38) is their ability to constantly cycle between the TGN and the cell surface (16Voorhoes P. Deignan E. van Donselaar E. Humphrey J. Marks M. Peters P.J. Bonifacino J.S. EMBO J. 1995; 14: 4961-4975Crossref PubMed Scopus (187) Google Scholar,18Bos K. Wraight C. Stanley K. EMBO J. 1993; 12: 2219-2228Crossref PubMed Scopus (193) Google Scholar, 31Molloy S.S. Thomas L. VanSlyke J.K. Stenberg P.E. Thomas G. EMBO J. 1994; 13: 18-33Crossref PubMed Scopus (420) Google Scholar). Since gI is found mainly in the TGN in the presence of gE, we wanted to investigate whether this also involves cycling of gI between these two compartments. To address this question, anti-gE and anti-gI antibody uptake experiments were performed on HeLa cells that had been double-transfected with gE and gI expression constructs. As shown in Fig. 2 c and in agreement with our previous findings (12Alconada A. Bauer U. Hoflack B. EMBO J. 1996; 15: 6096-6110Crossref PubMed Scopus (136) Google Scholar), after 1 h of incubation, the anti-gE antibodies were mainly concentrated in the perinuclear region of the cell, as a result of their internalization bound to the luminal domain of the recycling gE molecules. Interestingly, the anti-gI monoclonal antibody, when incubated with the cells for the same time, was also found in the perinuclear region, colocalizing with the anti-gE antibodies (Fig.2 d). In contrast, when the same experiment was performed on cells transfected exclusively with the pSFFV-gI construct, only cell surface bound anti-gI antibody could be detected (data not shown). These data indicate that gI, when simultaneously co-expressed with gE, cycles between the TGN and the cell surface, and that its accumulation in the TGN most likely depends on its rapid internalization from the cell surface together with gE, suggesting that the distribution of gI when co-expressed with gE is indistinguishable from that observed for gE when expressed alone. The results presented so far suggest that gE and gI are found within a complex in the cell whose traffic and distribution is solely determined by the sorting information in the cytoplasmic tail of gE. To verify this hypothesis, we constructed a modified version of gE in which its cytoplasmic tail had been replaced by that of the yeast protein Wbp1p, a type-I membrane protein that forms part of the ER resident oligosaccharyl-transferase complex (32Silberstein S. Gilmore R. FASEB J. 1996; 10: 849-858Crossref PubMed Scopus (207) Google Scholar). The tail of Wbp1p, which contains a consensus KKXX ER retention motif, has been shown to be sufficient to confer ER localization to reporter molecules both in mammalian and yeast cell systems (33Cosson P. Letorneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (483) Google Scholar, 34Letourneur F. Gaynor E.C. Demolliere C. Duden R. Emr S.D. Riezman H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (671) Google Scholar). When the gE-KKXX and the gI expression constructs were simultaneously transfected into HeLa cells and the localization of both molecules was assessed by indirect immunofluorescence, both the gE-KKXXchimera and gI were found in a cytoplasmic reticular compartment showing all the morphological features of the ER (Fig.3, a and b). As a control, we also constructed a gE-SSXX expression plasmid, in which the two lysines at positions −3 and −4 in the KKXX signal have been replaced by serines. This mutation is known to abolish the ER retention capacity of the KKXX motif (33Cosson P. Letorneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (483) Google Scholar). As expected, in cells co-expressing the gE-SSXX mutant and gI, both molecules were only detected at the cell surface (Fig. 3,c and d). These result confirms our prediction that the intracellular traffic of gI is exclusively determined by the sorting information on the tail of gE, presumably as a reflect of their association in the early secretory pathway. We then asked whether the expression of one given protein could influence the maturation of the other. In order to address this question, gE and gI were expressed either alone or simultaneously in HeLa cells with the help of a T7 RNA-polymerase recombinant vaccinia virus. The cells were metabolically labeled with radioactive methionine, chased for increasing periods of time, and lysed, and the lysates were immunoprecipitated with anti-gE- and anti-gI-specific antibodies. To identify the precursor and mature forms of gE and gI, both molecules were immunoprecipitated from cells lysates that were obtained either immediately after the labeling period or after 6 h of chase. The results showed that gE was initially synthesized as a 70-kDa band that was converted during the chase to a 100-kDa polypeptide, and gI was initially found as a 50-kDa band that matured to yield a fuzzy 65-kDa band (Fig.4 a). These values are in agreement with those found by other groups, either in transfected (21Yao Z. Jackson W. Forghani B. Grose C. J. Virol. 1993; 67: 305-314Crossref PubMed Google Scholar,35Litwin V. Jackson W. Grose C. J. Virol. 1992; 66: 3643-3651Crossref PubMed Google Scholar) or in VZV-infected cells (36Montalvo E.A. Parmley R.T. Grose C. J. Virol. 1985; 53: 761-770Crossref PubMed Google Scholar), for both the precursor and mature forms of gE and gI. When gE was expressed alone, immunoprecipitation with anti-gE antibodies revealed that maturation of the protein occurred rather rapidly, since as early as 20 min after initiation of the chase, almost 50% of the labeled 70-kDa precursor molecule was converted to the mature 100-kDa form (Fig. 4, b andc). When gI was expressed alone and immunoprecipitated with anti-gI antibodies under analogous conditions, its processing occurred very slowly, requiring more than 1 h to convert only 20% of the precursor to the mature form (Fig. 4, b and c). However, when gE and gI were expressed together, processing of gI was considerably enhanced, because 50% of the mature form could be detected after only 40 min of chase (Fig. 4, b andc). Under the same conditions, no difference was observed in the maturation of gE, when compared with the results obtained when this protein was expressed alone (Fig. 4 b). In addition, the anti-gE- and anti-gI-specific antibodies failed to immunoprecipitate any gI and gE, respectively (data not shown). It is worth mentioning that, when both proteins were expressed together, processing of gI occurred almost with identical kinetics as the processing of gE (Fig. 4 c). However, under these conditions, we reproducibly observed a decrease in the amounts of gE and gI that could be immunoprecipitated with their cognate antibodies when compared with the single transfections (Fig. 4 b). Since the expression l

We have studied the intracellular trafficking of the envelope glycoprotein I (gpI) of the varicella-zoster virus, a human herpes virus whose assembly is believed to occur in the trans-Golgi network (TGN) and/or in endocytic compartments. When expressed in HeLa cells in the absence of additional virally encoded factors, this type-I membrane protein localizes to the TGN and cycles between this compartment and the cell surface. The expression of gpI promotes the recruitment of the AP-1 Golgi-specific assembly proteins onto TGN membranes, strongly suggesting that gpI, like the mannose 6-phosphate receptors, can leave the TGN in clathrin-coated vesicles for subsequent transport to endosomes. Its return from the cell surface to the TGN also occurs through endosomes. The transfer of the gpI cytoplasmic domain onto a reporter molecule shows that this domain is sufficient to confer TGN localization. Mutational analysis of this domain indicates that proper subcellular localization and cycling of gpI depend on two different determinants, a tyrosine-containing tetrapeptide related to endocytosis sorting signals and a cluster of acidic amino acids containing casein kinase II phosphorylatable residues. Thus, the VZV gpI and the mannose 6-phosphate receptors, albeit localized in different intracellular compartments at steady-state, follow similar trafficking pathways and share similar sorting mechanisms.

The preprotein translocase of the outer mitochondrial membrane is a multi‐subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re‐establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.

Publisher Summary The identification of the mitochondrial receptor complex in yeast and N. crassa may be viewed as a paradigm of how biochemical approaches aimed at the study of protein–protein interactions can be successfully used for characterizing the different components of a protein complex, once the first component has been identified. Structural studies of the mitochondrial receptor complex will provide an enormous wealth of information on the molecular details involved in the interaction among the different components of the complex and between these components and the precursor proteins. The development of optical biosensor- based methodologies for studying protein–protein interactions will provide additional tools for this kind of molecular analysis. As the ultimate goal, the reconstitution of the fully functional translocation machinery into artificial lipid vesicles from purified complex components should provide detailed mechanistic information about the process of the translocation of precursor proteins into mitochondria.

The mitochondrial outer membrane contains import receptors for preproteins and a multisubunit general insertion pore. Several small outer membrane proteins (< 10 kDa) have been identified by their association with receptors or the general insertion pore, yet little is known about their function. Here, we present evidence that the biochemically identified Mom8b and the genetically identified Isp6 are identical. A deletion of Mom8b/Isp6 in Saccharomyces cerevisiae leads to (i) a delay of import of preproteins, (ii) stabilization of preprotein binding to receptors and the general insertion pore, and (iii) destabilization of the interaction between receptors and the general insertion pore. These results suggest that Mom8b supports the cooperativity between receptors and the general insertion pore and facilitates the release of preproteins from import components and thereby promotes efficient transfer of preproteins.

Antibodies raised against two synthetic peptides from rat liver F-1-ATPase alpha-subunit sequence recognized two main heat-shock proteins from Drosophila (p71 and p56) and rat liver (p74 and p54) cells. One of the antisera showed a 20-fold higher reactivity toward Escherichia coli GroEL chaperonin than toward the cu-subunit purified from Drosophila. Indirect immunofluorescence microscopy and subcellular fractionation experiments located both mammalian heat-shock proteins in the mitochondria. The recent findings of functional homology between chaperonins and alpha-subunits, together with the unsuspected immunological reactivity of two mitochondrial molecular chaperones toward antisera derived from two different sequence motifs of the alpha-subunit, strongly argue in favor of the existence of an evolutionary relationship between chaperonins and alpha-subunits. The complete sequence alignment of F-type ATPase alpha-subunits and chaperonins revealed the existence of eleven most conserved regions (similar to 30% of each protein sequence) with an overall amino acid identity of 20 +/- 2% and similarity of 39 +/- 4%. A search of protein data bases with three different consensus sequences derived from this alignment identified a significant proportion of proteins belonging only to these two protein families. Since the alpha-subunit protein family is evolutionary related to the other catalytic (A and beta) and regulatory (B) subunits of V- and F-type ATPases, the homology reported herein allowed us to analyze, in the chaperonin sequences, the conservation of critical residues involved in nucleotide binding. These data support the hypothesis that chaperonins and the major subunits of V- and F-type ATPases are evolutionary related.

A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F1-ATPase alpha-subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F1-ATPase alpha-subunit.

We report the one-step processing of the rat liver beta-F1-ATPase precursor protein, as examined by high resolution 2D-gel electrophoresis. Proteolytic cleavage of the positively charged mitochondrial targeting signal of the precursor promotes decreases in both the molecular weight (approximately 3 kDa) and the isoelectric point (approximate 0.2 pH unit) of the protein. The results obtained illustrate the usefulness of this technique, since it takes advantage of both results of the maturation process, for molecular characterization of the processing of mitochondrial precursor proteins.

Eukaryotic hsp60s are plastid-specific molecular chaperones implicated in the pathogenesis of many inflammatory and autoimmune diseases. We have used immunoelectron microscopy, immunoblotting and subcellular fractionation of islet cells to determine whether analogous proteins with related function are expressed in other cellular structures and whether such hsp60-related proteins could serve as antigenic targets in autoimmune diabetes. Using a panel of monoclonal and polyclonal antibodies to human and yeast hsp60s and immunoelectron microscopy, the hsp60 antibody cross-reactive proteins were detected in secretory granules, mitochondria, synaptic-like microvesicles and microtubules of mouse pancreatic beta cells. The expression of microtubule-associated hsp60 was induced by an infiltration of islets by mononuclear cells. This novel inducible-form of hsp60-related protein was recognized as an antigen by sera from diabetic mice. Subcellular fractionation of islets indicated that the molecular size of hsp60-related proteins included 66, 62, 58, 55, 52 and 38 kDa. These results demonstrate that the pancreatic beta cells express a family of hsp60-related proteins, with members differentially expressed in distinct cellular compartments. These proteins bearing hsp60 epitopes were antigenic targets for autoimmune responses in diabetic NOD mice.

The recent identification of the alpha-subunit of mitochondrial F1-ATPase complex in rat liver peroxisomes suggests another functional role for this protein in both organelles in addition to its involvement in mitochondrial oxidative phosphorylation. We report here that a very rapid response (15 min) in the induction of the alpha-regulatory subunit of the mitochondrial F1-ATPase complex is observed in 37 degrees C heat-shocked larvae of Drosophila hydei. Under the same heat-shock treatment, a similar-fold induction for the heat-shock protein hsp-70 was less rapid (45 min). Although the amino acid sequence identities between the "chaperonine" and the alpha-subunit protein families are very low (less than 20%), two amino acid sequences, of 12 and 13 residues each, are found in the alpha-subunits of the F1-ATPase complex from various eukaryotes which show a highly conserved identity (over 50%) with amino acid sequences found in molecular chaperones. We suggest that the nuclear coded alpha-subunit belongs to the family of stress proteins hsp-60 and thus, that it could perform similar functional role(s) to those recently described for mitochondrial hsp-60 (Cheng, M. Y., Hartl, F. U., Martin, J., Pollock, R. A., Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R. L., and Horwich, A. L. (1989) Nature 337, 620-625 and Ostermann, J., Horwich, A. L., Neupert, W., and Ultrich-Hartl, F. (1989) Nature 341, 125-130) in both the mitochondria and the peroxisomes. Furthermore, we suggest that the two conserved elements among the chaperonines and the alpha-subunits could putatively be involved in the chaperonine function of these proteins.

Postnatal structural, enzymatic, and bioenergetic development of mitochondria in the tissues of the newborn mammal is a key metabolic process necessary for adaptation to extra-uterine life. We have recently shown that postnatal mitochondrial differentiation in rat liver’ and brown adipose tissue2 is a rapid process that takes place during the first postnatal hour and depends on the increase in rates of synthesis for inner mitochondrial membrane proteins involved in respiration and oxidative phosphorylation. After birth until effective suckling begins, pyruvate is the main energy substrate oxidized by the tissues of the newborn rat.3 Irreversible oxidation of pyruvate to acetyl-CoA is catalyzed by mitochondria1 matrix-located pyruvate dehydrogenase complex (PDC) which contains multiple copies of three catalytic and two regulatory components and a protein X.4 Pyruvate oxidation depends on the activity of “active” PDC. “Active” PDC in newborn rat liver increases transiently during the first postnatal hour,’ whereas “total” PDC activity increases gradually over the entire neonatal period: Because of the physiological importance of the PDC in metabolic adaptation after birth and because mitochondrial differentiation has not been defined at the level of matrixlocated enzymes, the aim of the present investigation was to study the mechanism(s) responsible for the rapid postnatal increase of PDC activity in the newborn rat liver during the first postnatal hour. PDC activity was measured as previously described’ in freeze-clamped liver samples of newborn rats during the first 6 hr following birth. “Total” PDC activity was assayed after maximum dephosphorylation of the complex with an exogenously added partially purified phosphatase from pigeon acetone powder.’ FIGURE 1 shows that maximum activation of PDC activity was achieved by 20 min preincubation with this