Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunolocalization of p23 after microtubule disruption with nocodazole. HeLa  cells were treated with 10 μM nocodazole for 2 h  to induce the formation of dispersed Golgi  stacks, were fixed, and then processed for double immunofluorescence with antibodies against  p23 and ERD2, ERGIC-53, myc-NAGT I, or  TGN38. For double immunofluorescence of  p23 with ERD2 or TGN38, biotinylated CT antibody was revealed with streptavidin-FITC.  Fluorescein and rhodamine channels were  merged (overlay) after adjustment of both fluorescence signals to similar levels. The inset  shows a higher magnification of the area indicated by an arrowhead. The p23 signal overlapped completely with that of the KDEL receptor, ERD2, and extensively with that of  ERGIC-53. Numerous ERGIC-53–positive  membranes, corresponding to the ER, did not  contain p23. Segregation of NAGT I and p23  occurred in perinuclear Golgi fragments that  labeled for both markers, and in smaller peripheral structures that only labeled for p23.  The absence of signal overlap between p23 and  TGN38 was obvious. The colocalization of p23  with proteins of the cis-Golgi (ERD2), and the  segregation of p23 and proteins of the medial  (NAGT I) and trans-Golgi (TGN38) indicates  that p23 localizes to the cis side of the Golgi.  Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140216&req=5

Figure 3: Immunolocalization of p23 after microtubule disruption with nocodazole. HeLa cells were treated with 10 μM nocodazole for 2 h to induce the formation of dispersed Golgi stacks, were fixed, and then processed for double immunofluorescence with antibodies against p23 and ERD2, ERGIC-53, myc-NAGT I, or TGN38. For double immunofluorescence of p23 with ERD2 or TGN38, biotinylated CT antibody was revealed with streptavidin-FITC. Fluorescein and rhodamine channels were merged (overlay) after adjustment of both fluorescence signals to similar levels. The inset shows a higher magnification of the area indicated by an arrowhead. The p23 signal overlapped completely with that of the KDEL receptor, ERD2, and extensively with that of ERGIC-53. Numerous ERGIC-53–positive membranes, corresponding to the ER, did not contain p23. Segregation of NAGT I and p23 occurred in perinuclear Golgi fragments that labeled for both markers, and in smaller peripheral structures that only labeled for p23. The absence of signal overlap between p23 and TGN38 was obvious. The colocalization of p23 with proteins of the cis-Golgi (ERD2), and the segregation of p23 and proteins of the medial (NAGT I) and trans-Golgi (TGN38) indicates that p23 localizes to the cis side of the Golgi. Bar, 5 μm.

Mentions: To better investigate the distribution of p23, we used nocodazole. This drug causes microtubule depolymerization and the appearance of small Golgi stacks scattered throughout the cytoplasm (Rogalski and Singer, 1984; Cole et al., 1996). After depolymerization of microtubules with nocodazole (not shown), p23 appeared scattered throughout the cytoplasm (Fig. 3), as did all Golgi markers we tested (Fig. 3). The p23 membranes were positive for both ERD2 (Fig. 3, ERD2) and ERGIC-53 (Fig. 3, ERGIC-53). Whereas the distributions of p23 and ERD2 largely overlapped (Fig. 3, ERD2), numerous ERGIC-53–positive membranes, presumably derived from the ER, were negative for p23 (Fig. 3, ERGIC-53). Within the dispersed Golgi stacks, p23 was partially segregated from the medial-Golgi enzyme NAGT I (Fig. 3, NAGT I) and completely separated from the trans-Golgi protein TGN38 (Fig. 3, TGN38) (Luzio et al., 1990). The differential degree of colocalization of p23 with proteins of the Golgi and of the cis-Golgi/ intermediate compartment suggested that p23 localizes to the cis side of the Golgi apparatus.


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)

Immunolocalization of p23 after microtubule disruption with nocodazole. HeLa  cells were treated with 10 μM nocodazole for 2 h  to induce the formation of dispersed Golgi  stacks, were fixed, and then processed for double immunofluorescence with antibodies against  p23 and ERD2, ERGIC-53, myc-NAGT I, or  TGN38. For double immunofluorescence of  p23 with ERD2 or TGN38, biotinylated CT antibody was revealed with streptavidin-FITC.  Fluorescein and rhodamine channels were  merged (overlay) after adjustment of both fluorescence signals to similar levels. The inset  shows a higher magnification of the area indicated by an arrowhead. The p23 signal overlapped completely with that of the KDEL receptor, ERD2, and extensively with that of  ERGIC-53. Numerous ERGIC-53–positive  membranes, corresponding to the ER, did not  contain p23. Segregation of NAGT I and p23  occurred in perinuclear Golgi fragments that  labeled for both markers, and in smaller peripheral structures that only labeled for p23.  The absence of signal overlap between p23 and  TGN38 was obvious. The colocalization of p23  with proteins of the cis-Golgi (ERD2), and the  segregation of p23 and proteins of the medial  (NAGT I) and trans-Golgi (TGN38) indicates  that p23 localizes to the cis side of the Golgi.  Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140216&req=5

Figure 3: Immunolocalization of p23 after microtubule disruption with nocodazole. HeLa cells were treated with 10 μM nocodazole for 2 h to induce the formation of dispersed Golgi stacks, were fixed, and then processed for double immunofluorescence with antibodies against p23 and ERD2, ERGIC-53, myc-NAGT I, or TGN38. For double immunofluorescence of p23 with ERD2 or TGN38, biotinylated CT antibody was revealed with streptavidin-FITC. Fluorescein and rhodamine channels were merged (overlay) after adjustment of both fluorescence signals to similar levels. The inset shows a higher magnification of the area indicated by an arrowhead. The p23 signal overlapped completely with that of the KDEL receptor, ERD2, and extensively with that of ERGIC-53. Numerous ERGIC-53–positive membranes, corresponding to the ER, did not contain p23. Segregation of NAGT I and p23 occurred in perinuclear Golgi fragments that labeled for both markers, and in smaller peripheral structures that only labeled for p23. The absence of signal overlap between p23 and TGN38 was obvious. The colocalization of p23 with proteins of the cis-Golgi (ERD2), and the segregation of p23 and proteins of the medial (NAGT I) and trans-Golgi (TGN38) indicates that p23 localizes to the cis side of the Golgi. Bar, 5 μm.
Mentions: To better investigate the distribution of p23, we used nocodazole. This drug causes microtubule depolymerization and the appearance of small Golgi stacks scattered throughout the cytoplasm (Rogalski and Singer, 1984; Cole et al., 1996). After depolymerization of microtubules with nocodazole (not shown), p23 appeared scattered throughout the cytoplasm (Fig. 3), as did all Golgi markers we tested (Fig. 3). The p23 membranes were positive for both ERD2 (Fig. 3, ERD2) and ERGIC-53 (Fig. 3, ERGIC-53). Whereas the distributions of p23 and ERD2 largely overlapped (Fig. 3, ERD2), numerous ERGIC-53–positive membranes, presumably derived from the ER, were negative for p23 (Fig. 3, ERGIC-53). Within the dispersed Golgi stacks, p23 was partially segregated from the medial-Golgi enzyme NAGT I (Fig. 3, NAGT I) and completely separated from the trans-Golgi protein TGN38 (Fig. 3, TGN38) (Luzio et al., 1990). The differential degree of colocalization of p23 with proteins of the Golgi and of the cis-Golgi/ intermediate compartment suggested that p23 localizes to the cis side of the Golgi apparatus.

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