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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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p23 localizes to the intermediate compartment after  BFA treatment. HeLa cells were treated with 20 μg/ml BFA for 1 h  and fixed with methanol (a and b) or PFA (c–f). Cells were double labeled with antibodies against p23 (a, LP1; c and e, CT) and  ERGIC-53 (b), the KDEL receptor ERD2 (d) or myc-NAGT I  (f). Whereas the medial Golgi enzyme NAGT I relocated to the  ER (f), p23 appeared within discrete punctate structures scattered throughout the cytoplasm (a, c, and e), where it colocalized  (arrowheads) with ERGIC-53 (b) and ERD2 (d). Bar, 5 μm.
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Figure 5: p23 localizes to the intermediate compartment after BFA treatment. HeLa cells were treated with 20 μg/ml BFA for 1 h and fixed with methanol (a and b) or PFA (c–f). Cells were double labeled with antibodies against p23 (a, LP1; c and e, CT) and ERGIC-53 (b), the KDEL receptor ERD2 (d) or myc-NAGT I (f). Whereas the medial Golgi enzyme NAGT I relocated to the ER (f), p23 appeared within discrete punctate structures scattered throughout the cytoplasm (a, c, and e), where it colocalized (arrowheads) with ERGIC-53 (b) and ERD2 (d). Bar, 5 μm.

Mentions: The distribution of p23 suggested that the protein localizes to early biosynthetic membranes at the cis side of the Golgi complex. BFA causes COP I release from Golgi membranes, and the relocalization of Golgi proteins to the ER (Klausner et al., 1992). In contrast, ERGIC-53/p58, ERD2, and other proteins of the cis-Golgi/intermediate compartment (gp74, p210, and GM130), remain well separated from the ER after BFA treatment (Lippincot-Schwartz et al., 1990; Saraste and Svensson, 1991; Tang et al., 1993; Alcalde et al., 1994; Rios et al., 1994; Nakamura et al., 1995). After treatment of HeLa cells with BFA, the distribution of COP I proteins changed within minutes from the typical Golgi pattern to a cytoplasmic staining (not shown), and NAGT I was relocalized to the ER (Fig. 5 f). In contrast, p23 did not redistribute to the ER, but appeared within discrete punctate structures (Fig. 5, a, c, and e), which were also positive for both ERGIC-53 (Fig. 5 b) and ERD2 (Fig. 5 d). These observations confirmed that ERGIC-53 (Lippincott-Schwartz et al., 1990; Saraste and Svensson, 1991) and ERD2 (Tang et al., 1993; Scheel et al., 1997) do not redistribute to the ER upon BFA treatment. Changes in p23 distribution caused by BFA may indicate that the steady-state distribution of p23 relies on constant (re)cycling between early biosynthetic compartments, as has been observed for other proteins with a similar subcellular distribution (compare with Lippincott-Schwartz et al., 1990; Alcalde et al., 1994). Alternatively, it is also possible that changes in p23 distribution caused by BFA are due to drug-induced changes in membrane organization. More importantly, these experiments showed that p23 exhibited the typical behavior of cis-Golgi/intermediate compartment proteins after BFA treatment.


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)

p23 localizes to the intermediate compartment after  BFA treatment. HeLa cells were treated with 20 μg/ml BFA for 1 h  and fixed with methanol (a and b) or PFA (c–f). Cells were double labeled with antibodies against p23 (a, LP1; c and e, CT) and  ERGIC-53 (b), the KDEL receptor ERD2 (d) or myc-NAGT I  (f). Whereas the medial Golgi enzyme NAGT I relocated to the  ER (f), p23 appeared within discrete punctate structures scattered throughout the cytoplasm (a, c, and e), where it colocalized  (arrowheads) with ERGIC-53 (b) and ERD2 (d). Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 5: p23 localizes to the intermediate compartment after BFA treatment. HeLa cells were treated with 20 μg/ml BFA for 1 h and fixed with methanol (a and b) or PFA (c–f). Cells were double labeled with antibodies against p23 (a, LP1; c and e, CT) and ERGIC-53 (b), the KDEL receptor ERD2 (d) or myc-NAGT I (f). Whereas the medial Golgi enzyme NAGT I relocated to the ER (f), p23 appeared within discrete punctate structures scattered throughout the cytoplasm (a, c, and e), where it colocalized (arrowheads) with ERGIC-53 (b) and ERD2 (d). Bar, 5 μm.
Mentions: The distribution of p23 suggested that the protein localizes to early biosynthetic membranes at the cis side of the Golgi complex. BFA causes COP I release from Golgi membranes, and the relocalization of Golgi proteins to the ER (Klausner et al., 1992). In contrast, ERGIC-53/p58, ERD2, and other proteins of the cis-Golgi/intermediate compartment (gp74, p210, and GM130), remain well separated from the ER after BFA treatment (Lippincot-Schwartz et al., 1990; Saraste and Svensson, 1991; Tang et al., 1993; Alcalde et al., 1994; Rios et al., 1994; Nakamura et al., 1995). After treatment of HeLa cells with BFA, the distribution of COP I proteins changed within minutes from the typical Golgi pattern to a cytoplasmic staining (not shown), and NAGT I was relocalized to the ER (Fig. 5 f). In contrast, p23 did not redistribute to the ER, but appeared within discrete punctate structures (Fig. 5, a, c, and e), which were also positive for both ERGIC-53 (Fig. 5 b) and ERD2 (Fig. 5 d). These observations confirmed that ERGIC-53 (Lippincott-Schwartz et al., 1990; Saraste and Svensson, 1991) and ERD2 (Tang et al., 1993; Scheel et al., 1997) do not redistribute to the ER upon BFA treatment. Changes in p23 distribution caused by BFA may indicate that the steady-state distribution of p23 relies on constant (re)cycling between early biosynthetic compartments, as has been observed for other proteins with a similar subcellular distribution (compare with Lippincott-Schwartz et al., 1990; Alcalde et al., 1994). Alternatively, it is also possible that changes in p23 distribution caused by BFA are due to drug-induced changes in membrane organization. More importantly, these experiments showed that p23 exhibited the typical behavior of cis-Golgi/intermediate compartment proteins after BFA treatment.

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