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Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Rojo M, Pepperkok R, Emery G, Kellner R, Stang E, Parton RG, Gruenberg J - J. Cell Biol. (1997)

Bottom Line: Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment.Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules.These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

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Cofractionation of p23  and ERD2. BHK cells were homogenized and membranes of  the postnuclear supernatant  were separated in a step sucrose  gradient. In a, fractions were analyzed by Western blot (equal  amounts of protein loaded per  lane), and antibodies were revealed by chemiluminescence.  In b, the protein content of each  fraction is expressed as a percentage of the total amount in  the gradient. The p23 protein  and ERD2 were enriched in the  M2 interface. Cytosolic proteins, including the bulk of β-COP  (a), remained in the load of the gradient (b, 40%).
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Figure 7: Cofractionation of p23 and ERD2. BHK cells were homogenized and membranes of the postnuclear supernatant were separated in a step sucrose gradient. In a, fractions were analyzed by Western blot (equal amounts of protein loaded per lane), and antibodies were revealed by chemiluminescence. In b, the protein content of each fraction is expressed as a percentage of the total amount in the gradient. The p23 protein and ERD2 were enriched in the M2 interface. Cytosolic proteins, including the bulk of β-COP (a), remained in the load of the gradient (b, 40%).

Mentions: Immunofluorescence with anti-p23 antibodies revealed perinuclear structures with a Golgi-like aspect in different cell types (Fig. 2, BHK and HeLa; see Fig. 7, CHO and Vero; MDCK, not shown). Similar patterns were observed using two different antibodies, CT and LP1, and two different fixation protocols (PFA and methanol). This distribution closely resembled that of rabbit p23 (Sohn et al., 1996). As shown in Fig. 2, p23 overlapped with NAGT I (Fig. 2, a and b) and the KDEL-receptor ERD2 (Fig. 2, c and d). Whereas NAGT I is restricted to medial Golgi cisternae (Nilsson et al., 1993), the KDEL receptor, ERD2, has been localized both to the Golgi stack and to the intermediate compartment between ER and Golgi (Tang et al., 1993; Griffiths et al., 1994). In addition, p23 partially overlapped with ERGIC-53, but only within the perinuclear Golgi area (Fig. 2, e and f). Indeed, ERGIC-53 is used as a marker of the intermediate compartment (Schweizer et al., 1988, 1990), but the protein cycles between ER and Golgi (Lippincott-Schwartz et al., 1990) and a significant fraction of ERGIC-53 molecules localize to the ER at steady state in several cell types (Hans P. Hauri, personal communication) (Fig. 2 f, HeLa cells).


Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Rojo M, Pepperkok R, Emery G, Kellner R, Stang E, Parton RG, Gruenberg J - J. Cell Biol. (1997)

Cofractionation of p23  and ERD2. BHK cells were homogenized and membranes of  the postnuclear supernatant  were separated in a step sucrose  gradient. In a, fractions were analyzed by Western blot (equal  amounts of protein loaded per  lane), and antibodies were revealed by chemiluminescence.  In b, the protein content of each  fraction is expressed as a percentage of the total amount in  the gradient. The p23 protein  and ERD2 were enriched in the  M2 interface. Cytosolic proteins, including the bulk of β-COP  (a), remained in the load of the gradient (b, 40%).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140216&req=5

Figure 7: Cofractionation of p23 and ERD2. BHK cells were homogenized and membranes of the postnuclear supernatant were separated in a step sucrose gradient. In a, fractions were analyzed by Western blot (equal amounts of protein loaded per lane), and antibodies were revealed by chemiluminescence. In b, the protein content of each fraction is expressed as a percentage of the total amount in the gradient. The p23 protein and ERD2 were enriched in the M2 interface. Cytosolic proteins, including the bulk of β-COP (a), remained in the load of the gradient (b, 40%).
Mentions: Immunofluorescence with anti-p23 antibodies revealed perinuclear structures with a Golgi-like aspect in different cell types (Fig. 2, BHK and HeLa; see Fig. 7, CHO and Vero; MDCK, not shown). Similar patterns were observed using two different antibodies, CT and LP1, and two different fixation protocols (PFA and methanol). This distribution closely resembled that of rabbit p23 (Sohn et al., 1996). As shown in Fig. 2, p23 overlapped with NAGT I (Fig. 2, a and b) and the KDEL-receptor ERD2 (Fig. 2, c and d). Whereas NAGT I is restricted to medial Golgi cisternae (Nilsson et al., 1993), the KDEL receptor, ERD2, has been localized both to the Golgi stack and to the intermediate compartment between ER and Golgi (Tang et al., 1993; Griffiths et al., 1994). In addition, p23 partially overlapped with ERGIC-53, but only within the perinuclear Golgi area (Fig. 2, e and f). Indeed, ERGIC-53 is used as a marker of the intermediate compartment (Schweizer et al., 1988, 1990), but the protein cycles between ER and Golgi (Lippincott-Schwartz et al., 1990) and a significant fraction of ERGIC-53 molecules localize to the ER at steady state in several cell types (Hans P. Hauri, personal communication) (Fig. 2 f, HeLa cells).

Bottom Line: Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment.Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules.These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

Show MeSH
Related in: MedlinePlus