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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 block ER to Golgi transport of VSV-G protein at a pre-Golgi stage. NRK cells were infected with VSVtsO45 for 3 h at 42°C. Cells were permeabilized and supplemented with complete transport cocktail supplemented either with anti–p115 antibodies (A–C) or control antibodies (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti-p115 (A and D) and anti–VSV-G protein (B and E) antibodies. Addition of anti–p115 antibodies to the transport assay has no effect on VSV-G protein exit from the ER, but prevents VSV-G protein transport to the Golgi (A–C) and causes accumulation of VSV-G protein in scattered VTCs. Arrowheads point to peripheral structures containing VSV-G protein and anti–p115 antibodies. Arrows indicate Golgi elements labeled with anti–p115 antibodies but lacking VSV-G protein. Addition of control antibodies to the transport assay had no effect on VSV-G protein transport, and VSV-G protein was efficiently delivered to the Golgi (D–F). Bar 10 μm.
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Figure 7: Antibodies against p115 block ER to Golgi transport of VSV-G protein at a pre-Golgi stage. NRK cells were infected with VSVtsO45 for 3 h at 42°C. Cells were permeabilized and supplemented with complete transport cocktail supplemented either with anti–p115 antibodies (A–C) or control antibodies (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti-p115 (A and D) and anti–VSV-G protein (B and E) antibodies. Addition of anti–p115 antibodies to the transport assay has no effect on VSV-G protein exit from the ER, but prevents VSV-G protein transport to the Golgi (A–C) and causes accumulation of VSV-G protein in scattered VTCs. Arrowheads point to peripheral structures containing VSV-G protein and anti–p115 antibodies. Arrows indicate Golgi elements labeled with anti–p115 antibodies but lacking VSV-G protein. Addition of control antibodies to the transport assay had no effect on VSV-G protein transport, and VSV-G protein was efficiently delivered to the Golgi (D–F). Bar 10 μm.

Mentions: To define where anti–p115 antibodies and p115 depletion blocked transport of VSV-G protein, morphological transport assays were performed. Addition of anti–p115 antibodies (∼10 ng IgG/μl transport assay, an amount analogous to that shown to inhibit the biochemical assay in Fig. 5 A, lane 5) to the morphological transport assay prevented VSV-G protein from moving to the Golgi (Fig. 7, A–C). A representative experiment (from >10 analyses) is presented. VSV-G protein was not detected in the ER, indicating that anti–p115 antibodies had no effect on VSV-G protein exit from the ER and its delivery to post-ER transport intermediates, but prevented delivery of such intermediates to the Golgi. This resulted in accumulation of VSV-G protein in peripheral VTCs, most of which were labeled with anti–p115 antibodies (C, arrowheads). Anti–p115 antibodies were also detected in the Golgi (C, arrows), perhaps because of incomplete removal of p115 from membranes during permeabilization. Alternatively, anti–p115 antibodies might act by trapping p115 on Golgi membranes through the formation of inactive complexes. Addition of equivalent amounts of preimmune antibodies to the transport assay had no effect on VSV-G protein transport, and VSV-G protein was efficiently delivered to the Golgi, where it colocalized with p115 (D–F). The same pattern was observed when monoclonal anti–Mann II antibodies were added to the reaction (data not shown).


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 block ER to Golgi transport of VSV-G protein at a pre-Golgi stage. NRK cells were infected with VSVtsO45 for 3 h at 42°C. Cells were permeabilized and supplemented with complete transport cocktail supplemented either with anti–p115 antibodies (A–C) or control antibodies (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti-p115 (A and D) and anti–VSV-G protein (B and E) antibodies. Addition of anti–p115 antibodies to the transport assay has no effect on VSV-G protein exit from the ER, but prevents VSV-G protein transport to the Golgi (A–C) and causes accumulation of VSV-G protein in scattered VTCs. Arrowheads point to peripheral structures containing VSV-G protein and anti–p115 antibodies. Arrows indicate Golgi elements labeled with anti–p115 antibodies but lacking VSV-G protein. Addition of control antibodies to the transport assay had no effect on VSV-G protein transport, and VSV-G protein was efficiently delivered to the Golgi (D–F). Bar 10 μm.
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Related In: Results  -  Collection

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Figure 7: Antibodies against p115 block ER to Golgi transport of VSV-G protein at a pre-Golgi stage. NRK cells were infected with VSVtsO45 for 3 h at 42°C. Cells were permeabilized and supplemented with complete transport cocktail supplemented either with anti–p115 antibodies (A–C) or control antibodies (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti-p115 (A and D) and anti–VSV-G protein (B and E) antibodies. Addition of anti–p115 antibodies to the transport assay has no effect on VSV-G protein exit from the ER, but prevents VSV-G protein transport to the Golgi (A–C) and causes accumulation of VSV-G protein in scattered VTCs. Arrowheads point to peripheral structures containing VSV-G protein and anti–p115 antibodies. Arrows indicate Golgi elements labeled with anti–p115 antibodies but lacking VSV-G protein. Addition of control antibodies to the transport assay had no effect on VSV-G protein transport, and VSV-G protein was efficiently delivered to the Golgi (D–F). Bar 10 μm.
Mentions: To define where anti–p115 antibodies and p115 depletion blocked transport of VSV-G protein, morphological transport assays were performed. Addition of anti–p115 antibodies (∼10 ng IgG/μl transport assay, an amount analogous to that shown to inhibit the biochemical assay in Fig. 5 A, lane 5) to the morphological transport assay prevented VSV-G protein from moving to the Golgi (Fig. 7, A–C). A representative experiment (from >10 analyses) is presented. VSV-G protein was not detected in the ER, indicating that anti–p115 antibodies had no effect on VSV-G protein exit from the ER and its delivery to post-ER transport intermediates, but prevented delivery of such intermediates to the Golgi. This resulted in accumulation of VSV-G protein in peripheral VTCs, most of which were labeled with anti–p115 antibodies (C, arrowheads). Anti–p115 antibodies were also detected in the Golgi (C, arrows), perhaps because of incomplete removal of p115 from membranes during permeabilization. Alternatively, anti–p115 antibodies might act by trapping p115 on Golgi membranes through the formation of inactive complexes. Addition of equivalent amounts of preimmune antibodies to the transport assay had no effect on VSV-G protein transport, and VSV-G protein was efficiently delivered to the Golgi, where it colocalized with p115 (D–F). The same pattern was observed when monoclonal anti–Mann II antibodies were added to the reaction (data not shown).

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