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ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage.

Alvarez C, Fujita H, Hubbard A, Sztul E - J. Cell Biol. (1999)

Bottom Line: Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C.Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack.This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

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Antibodies against p115 cause Golgi complex disassembly. mAbs against p115 mixed with TR-dextran (A–C), or mAbs against 5′-nucleotidase mixed with TR-dextran (D–F) were microinjected into the cytoplasm of WIF-B cells. Phase images of cells are shown (A and D). Injected cells are identified by TR-dextran (B and E) and are traced in outline in C and F. Cells were fixed 2 h after injection and processed for immunofluorescence using antibodies against Mann II (C and F). Cells injected with anti–p115 antibodies, but not uninjected cells or cells injected with control antibodies show disassembly of Golgi complexes. Bar, 10 μm.
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Figure 1: Antibodies against p115 cause Golgi complex disassembly. mAbs against p115 mixed with TR-dextran (A–C), or mAbs against 5′-nucleotidase mixed with TR-dextran (D–F) were microinjected into the cytoplasm of WIF-B cells. Phase images of cells are shown (A and D). Injected cells are identified by TR-dextran (B and E) and are traced in outline in C and F. Cells were fixed 2 h after injection and processed for immunofluorescence using antibodies against Mann II (C and F). Cells injected with anti–p115 antibodies, but not uninjected cells or cells injected with control antibodies show disassembly of Golgi complexes. Bar, 10 μm.

Mentions: The requirement for p115 has been previously studied only in in vitro–reconstituted assays (Waters et al. 1992; Barroso et al. 1995; Rabouille et al. 1995, Rabouille et al. 1998). To examine the function of p115 in vivo, monoclonal anti–p115 antibodies were microinjected into WIF-B cells (Shanks et al. 1994), and their effect on Golgi structure was monitored by the distribution of Mann II. As shown in Fig. 1, injection of anti–p115 antibodies had an effect on Golgi structure. A field of cells is shown by phase-contrast (A), and the injected cells are identified by their content of coinjected TR-dextran (B). Within 2 h after injection, the Golgi complexes in all injected cells were disassembled, and Mann II was present in relatively large punctate structures dispersed throughout the cells (C). The structures appeared superficially similar to those observed in cells transfected with mutant forms of p115 lacking portions of the globular head or the coiled-coil tail (Nelson et al. 1998). Injection of control antibodies against the bile canalicular plasma membrane protein 5′-nucleotidase did not perturb Golgi structure (D–F).


ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage.

Alvarez C, Fujita H, Hubbard A, Sztul E - J. Cell Biol. (1999)

Antibodies against p115 cause Golgi complex disassembly. mAbs against p115 mixed with TR-dextran (A–C), or mAbs against 5′-nucleotidase mixed with TR-dextran (D–F) were microinjected into the cytoplasm of WIF-B cells. Phase images of cells are shown (A and D). Injected cells are identified by TR-dextran (B and E) and are traced in outline in C and F. Cells were fixed 2 h after injection and processed for immunofluorescence using antibodies against Mann II (C and F). Cells injected with anti–p115 antibodies, but not uninjected cells or cells injected with control antibodies show disassembly of Golgi complexes. Bar, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168100&req=5

Figure 1: Antibodies against p115 cause Golgi complex disassembly. mAbs against p115 mixed with TR-dextran (A–C), or mAbs against 5′-nucleotidase mixed with TR-dextran (D–F) were microinjected into the cytoplasm of WIF-B cells. Phase images of cells are shown (A and D). Injected cells are identified by TR-dextran (B and E) and are traced in outline in C and F. Cells were fixed 2 h after injection and processed for immunofluorescence using antibodies against Mann II (C and F). Cells injected with anti–p115 antibodies, but not uninjected cells or cells injected with control antibodies show disassembly of Golgi complexes. Bar, 10 μm.
Mentions: The requirement for p115 has been previously studied only in in vitro–reconstituted assays (Waters et al. 1992; Barroso et al. 1995; Rabouille et al. 1995, Rabouille et al. 1998). To examine the function of p115 in vivo, monoclonal anti–p115 antibodies were microinjected into WIF-B cells (Shanks et al. 1994), and their effect on Golgi structure was monitored by the distribution of Mann II. As shown in Fig. 1, injection of anti–p115 antibodies had an effect on Golgi structure. A field of cells is shown by phase-contrast (A), and the injected cells are identified by their content of coinjected TR-dextran (B). Within 2 h after injection, the Golgi complexes in all injected cells were disassembled, and Mann II was present in relatively large punctate structures dispersed throughout the cells (C). The structures appeared superficially similar to those observed in cells transfected with mutant forms of p115 lacking portions of the globular head or the coiled-coil tail (Nelson et al. 1998). Injection of control antibodies against the bile canalicular plasma membrane protein 5′-nucleotidase did not perturb Golgi structure (D–F).

Bottom Line: Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C.Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack.This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

Show MeSH
Related in: MedlinePlus