Localization and recycling of gp27 (hp24gamma3): complex formation with other p24 family members - PubMed (original) (raw)

Localization and recycling of gp27 (hp24gamma3): complex formation with other p24 family members

J Füllekrug et al. Mol Biol Cell. 1999 Jun.

Free PMC article

Abstract

We report here the characterization of gp27 (hp24gamma3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15 degrees C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24alpha2), p24 (hp24beta1), and p23 (hp24delta1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24gamma4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.

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Figures

Figure 1

Figure 1

Comparison between gp27 and Golgi marker proteins. Double immunofluorescence stainings of Vero cells were analyzed by confocal laser scanning microscopy. Marker proteins were chosen to represent subcompartments of the Golgi apparatus: KDEL receptor for CGN and _cis_-Golgi, NAGT I for medial-Golgi, and GalT (GTL2 antibody) for _trans_-Golgi and TGN. Note that the localization of KDEL receptor and gp27 is very similar, although intensities are different. NAGT I and, even more, GalT show Golgi patterns shifted against gp27. Bar, 10 μm.

Figure 2

Figure 2

Ultrastructural localization of gp27 to the cis side of the Golgi apparatus. (a) Single labeling of HeLa cells with gp27 followed by protein A-colloidal gold (10 nm). Golgi stacks (GS) usually comprise three cisternae, and labeling for gp27 is highly polarized. (b) Comparison between the _trans_-Golgi marker protein GalT (10 nm, rabbit antiserum N10) and gp27 (5 nm). Segregation between these two proteins identifies the gp27-containing cisternae as _cis_-Golgi. Bar, 100 nm.

Figure 3

Figure 3

Localization of gp27 to coated transport structures between ER and Golgi. Double immunofluorescence stainings of Vero cells were analyzed by confocal laser-scanning microscopy. Sec13/COPII and β′-COP/COPI show some coincident staining with gp27, which is more evident in the insets. For comparison, the much more obvious closely associated staining between COPII and COPI is shown in the lower panel. Bar, 10 μm.

Figure 4

Figure 4

Accumulation of gp27 in the ER–Golgi 15°C compartment. Vero cells were infected with VSV-Gts045 and kept for 3 h at the restrictive temperature. After addition of the protein synthesis inhibitor cycloheximide, cells were further incubated for 1 h at 15°C. Peripheral structures of gp27 grew in number and apparent size. Note the high degree of coincident staining with VSV-Gts045 glycoprotein arrested on its way from ER to the Golgi apparatus. Bar, 10 μm.

Figure 5

Figure 5

Subcellular distribution of gp27 after brefeldin A treatment. Resident glycosylation enzymes such as NAGT I stain nuclear envelope and reticular cytoplasmic ER structures, but gp27 distribution is different. A striking coincidence of staining is observed with KDEL receptor, and many of the brefeldin A–induced punctated structures are coincident with Sec13/COPII. Bar, 10 μm.

Figure 6

Figure 6

Microinjection of Sar1pdn to block ER export leads to a slow accumulation by 3 h of gp27 in the ER. HeLa cells were microinjected with Sar1pdn in the presence of 5 μg/ml emetine to inhibit protein synthesis. Cells were then incubated for 3 h at 37°C in complete media in the presence of emetine. Cells were fixed with formaldehyde and immunofluorescently labeled for gp27. The arrowhead points to the microinjected cell in this example field. Note that a juxtanuclear gp27 staining pattern of noninjected control cells is largely absent in the microinjected cell. Considerable diffuse cytoplasmic fluorescence indicative of ER localization is present in the injected cell. Bar, 10 μm.

Figure 7

Figure 7

Coimmunoprecipitation of p24 family proteins with gp27 affinity-purified antibodies. [35S]Methionine was used for labeling HeLa cells overnight, and lysis was routinely done with the nonionic detergent TX-100. Under these conditions, gp27 was efficiently solubilized (a, inset; p, pellet; S, supernatant), whereas large detergent-resistant structures containing caveolin, a protein residing mostly in the CGN and the _cis_-Golgi as well as on plasma membrane (Dupree et al., 1993; Parton, personal communication), were sedimented and would not be present in the starting lysate for immunoprecipitation. Immunoprecipitates obtained with gp27 antibodies were analyzed by 1D SDS-PAGE (a, lanes SDS and TX-100) and isoelectric focusing followed by SDS-PAGE (a–c). Under denaturing conditions (a, SDS), one prominent band at 30 kDa corresponding to gp27 is resolved. Using TX-100 (b, Tx-100), three major bands in the 20- to 30-kDa range were observed (A–C). 2D gel analysis resolved this pattern into five different spots: A1, A2, B, C1, and C2 (a). The same 2D gel loaded with 35S-labeled immunoprecipitate and unlabeled Golgi-enriched subcellular fraction is shown in a and b. After detection of radioactively labeled proteins (a), the membrane was probed with antibodies against p24 family proteins (b). Alignment of the two patterns identified coprecipitated proteins as different p24 family members. Protein A-peroxidase was applied for detection of primary antibodies against p24 family proteins, which also bound to IgGs of the gp27 antibodies used for immunoprecipitation, giving rise to the additional strong staining patterns apparent in b. Some additional spots of higher molecular mass are also apparent and are likely to be unspecific contaminants, because they are also seen when using preimmune serum (pI) or rabbit IgG (a, inset). 2D gels loaded with labeled immunoprecipitate without added Golgi fraction were of sufficient resolution to enable quantification (c).

Figure 8

Figure 8

Coexpression of p24 family proteins allows transport of gp27 from ER to the Golgi apparatus. HeLa cells were transiently transfected with gp27 cDNA (20%) and other p24 family cDNAs (20% each, pcDNA3 vector DNA making up to 100%). The Golgi apparatus was identified by mAb GTL2 against GalT. Without coexpressed p24 proteins, the predominant staining pattern for overexpressed gp27 is ER. Double transfection of gp27 with other p24 family cDNAs gives different phenotypes depending on which particular cDNA is used. In terms of Golgi localization, coexpression of p23, p24, and p25 is the most efficient combination. See table and text for details. The localization indicated in parentheses in the table is not the predominant phenotype but was observed consistently. Bar, 10 μm.

Figure 9

Figure 9

Glycosylation analysis of gp27 and GMP25 in HeLa cells. (a) Removal of N-linked oligosaccharides by peptide _N_-glycanase F led to faster migration of both proteins, but p26 is not affected. Maturation of the oligosaccharide chain of gp27 in the medial-Golgi renders it resistant to digestion with Endo H. The amount of Endo H–sensitive GMP25 is ≥95%. (b) Pulse–chase analysis of the acquisition of Endo H resistance for gp27. After 60 min, two-thirds of gp27 are already Endo H resistant.

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References

    1. Aridor M, Bannykh SI, Rowe T, Balch WE. Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport. J Cell Biol. 1995;131:875–893. - PMC - PubMed
    1. Belden WJ, Barlowe C. Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J Biol Chem. 1996;271:26939–26946. - PubMed
    1. Blum R, Feick P, Puype M, Vandekerckhove J, Klengel R, Nastainczyk W, Schulz I. Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking. J Biol Chem. 1996;271:17183–17189. - PubMed
    1. Dominguez M, Dejgaard K, Füllekrug J, Dahan S, Fazel A, Paccaud JP, Thomas DY, Bergeron JJ, Nilsson T. gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COP I and II coatomer. J Cell Biol. 1998;140:751–765. - PMC - PubMed
    1. Dunphy WG, Brands R, Rothman JE. Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell. 1985;40:463–472. - PubMed

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