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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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COP I and p23 colocalize in the intermediate compartment at 15°C, and segregate upon release of the  temperature block. Vero cells  were infected with VSV tsO45  and tsO45-G was blocked in  the intermediate compartment for 3 h at 15°C as described in Fig. 6. The temperature was shifted to 31°C for the  indicated periods of time, and  cells were processed for double immunofluorescence with  antibodies against p23 (CT)  and COP I. Images were processed and merged (overlay) as  in Fig. 3. The inset shows a  higher magnification of the  area indicated by an arrowhead. COP I and p23 colocalize in the intermediate compartment at 15°C (0′). Upon  release of the temperature  block (3′ and 6′), small COP  I–positive structures, that may  represent forward transport  vesicles, are devoid of p23.  Bar, 5 μm.
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Figure 9: COP I and p23 colocalize in the intermediate compartment at 15°C, and segregate upon release of the temperature block. Vero cells were infected with VSV tsO45 and tsO45-G was blocked in the intermediate compartment for 3 h at 15°C as described in Fig. 6. The temperature was shifted to 31°C for the indicated periods of time, and cells were processed for double immunofluorescence with antibodies against p23 (CT) and COP I. Images were processed and merged (overlay) as in Fig. 3. The inset shows a higher magnification of the area indicated by an arrowhead. COP I and p23 colocalize in the intermediate compartment at 15°C (0′). Upon release of the temperature block (3′ and 6′), small COP I–positive structures, that may represent forward transport vesicles, are devoid of p23. Bar, 5 μm.

Mentions: The effects of the “15°C block” are fully reversible and membrane traffic is re-established after shift back to 31°C (Saraste and Kuismanen, 1984). After release from the 15°C block, it is thus possible to analyze and compare the transport routes of different proteins (Griffiths et al., 1995; Tang et al., 1995). After accumulation of tsO45-G at 15°C, cells were shifted to 31°C for different times, and then analyzed by immunofluorescence. Within 3 min after shift, numerous vesicles containing tsO45-G were observed, presumably corresponding to transport intermediates carrying tsO45-G towards the Golgi complex (Fig. 6, 3′). Strikingly, these vesicles were devoid of p23 labeling, suggesting that p23 is absent from vesicles that carry tsO45-G beyond the intermediate compartment (see also Fig. 9). Between 12 and 30 min, the number of peripheral structures containing p23 but not ts045-G, probably corresponding to elements of the cis-Golgi and/or intermediate compartment, increased (Fig. 6, 12′ and 30′). During the same time both labels tended to overlap in the crescent of perinuclear Golgi membranes. This overlap may not reflect p23 and tsO45-G colocalization, but the inability to discriminate between two close but separared fluorescent signals, as was observed for the steady-state distribution of cis and medial Golgi markers (Fig. 2). Beyond 30 min, little if any colocalization of the two proteins could be observed (Fig. 6, 60′), while the bulk of tsO45-G had reached the plasma membrane (not visible in the confocal planes shown in Fig. 6). Altogether, these data indicate that p23 localizes to membranes that have been defined as intermediate compartment after BFA treatment or low (15°C) temperature incubations.


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)

COP I and p23 colocalize in the intermediate compartment at 15°C, and segregate upon release of the  temperature block. Vero cells  were infected with VSV tsO45  and tsO45-G was blocked in  the intermediate compartment for 3 h at 15°C as described in Fig. 6. The temperature was shifted to 31°C for the  indicated periods of time, and  cells were processed for double immunofluorescence with  antibodies against p23 (CT)  and COP I. Images were processed and merged (overlay) as  in Fig. 3. The inset shows a  higher magnification of the  area indicated by an arrowhead. COP I and p23 colocalize in the intermediate compartment at 15°C (0′). Upon  release of the temperature  block (3′ and 6′), small COP  I–positive structures, that may  represent forward transport  vesicles, are devoid of p23.  Bar, 5 μm.
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Figure 9: COP I and p23 colocalize in the intermediate compartment at 15°C, and segregate upon release of the temperature block. Vero cells were infected with VSV tsO45 and tsO45-G was blocked in the intermediate compartment for 3 h at 15°C as described in Fig. 6. The temperature was shifted to 31°C for the indicated periods of time, and cells were processed for double immunofluorescence with antibodies against p23 (CT) and COP I. Images were processed and merged (overlay) as in Fig. 3. The inset shows a higher magnification of the area indicated by an arrowhead. COP I and p23 colocalize in the intermediate compartment at 15°C (0′). Upon release of the temperature block (3′ and 6′), small COP I–positive structures, that may represent forward transport vesicles, are devoid of p23. Bar, 5 μm.
Mentions: The effects of the “15°C block” are fully reversible and membrane traffic is re-established after shift back to 31°C (Saraste and Kuismanen, 1984). After release from the 15°C block, it is thus possible to analyze and compare the transport routes of different proteins (Griffiths et al., 1995; Tang et al., 1995). After accumulation of tsO45-G at 15°C, cells were shifted to 31°C for different times, and then analyzed by immunofluorescence. Within 3 min after shift, numerous vesicles containing tsO45-G were observed, presumably corresponding to transport intermediates carrying tsO45-G towards the Golgi complex (Fig. 6, 3′). Strikingly, these vesicles were devoid of p23 labeling, suggesting that p23 is absent from vesicles that carry tsO45-G beyond the intermediate compartment (see also Fig. 9). Between 12 and 30 min, the number of peripheral structures containing p23 but not ts045-G, probably corresponding to elements of the cis-Golgi and/or intermediate compartment, increased (Fig. 6, 12′ and 30′). During the same time both labels tended to overlap in the crescent of perinuclear Golgi membranes. This overlap may not reflect p23 and tsO45-G colocalization, but the inability to discriminate between two close but separared fluorescent signals, as was observed for the steady-state distribution of cis and medial Golgi markers (Fig. 2). Beyond 30 min, little if any colocalization of the two proteins could be observed (Fig. 6, 60′), while the bulk of tsO45-G had reached the plasma membrane (not visible in the confocal planes shown in Fig. 6). Altogether, these data indicate that p23 localizes to membranes that have been defined as intermediate compartment after BFA treatment or low (15°C) temperature incubations.

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