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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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p115 depletion blocks 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 transport cocktails containing p115-depleted cytosol (A–C) or complete cytosol (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti–VSV-G protein (A and D) and anti–Mann II (B and E) antibodies. Depletion of p115 from the transport assay had no effect on VSV-G protein exit from the ER, but prevented VSV-G protein transport to the Golgi (A–C) and caused accumulation of VSV-G protein in peripheral VTCs lacking Mann II. In the presence of complete cytosol, VSV-G protein was efficiently delivered to the Golgi (D–F). Bar 10 μm.
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Figure 8: p115 depletion blocks 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 transport cocktails containing p115-depleted cytosol (A–C) or complete cytosol (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti–VSV-G protein (A and D) and anti–Mann II (B and E) antibodies. Depletion of p115 from the transport assay had no effect on VSV-G protein exit from the ER, but prevented VSV-G protein transport to the Golgi (A–C) and caused accumulation of VSV-G protein in peripheral VTCs lacking Mann II. In the presence of complete cytosol, VSV-G protein was efficiently delivered to the Golgi (D–F). Bar 10 μm.

Mentions: When p115-depleted cytosol (analogous to that unable to support the biochemical assay in Fig. 5 C, lane 4) was used in the morphological transport assay, VSV-G protein was also not transported to the Golgi, as evidenced by its lack of colocalization with Mann II (Fig. 8, A–C). Two representative cells from two different experiments are shown in these panels. VSV-G protein localized predominantly to peripheral VTCs, indistinguishable from those seen in the presence of anti–p115 antibodies (compare to Fig. 7 B). VSV-G protein was almost exclusively in peripheral VTCs, and was not present to any significant extent in the ER, confirming that p115 is not involved in ER exit and delivery of cargo to more distal transport intermediates. VSV-G protein was efficiently transported to the Golgi when complete cytosol was used (Fig. 8, D–F). Taken together, these results indicate that p115 function is required after the formation of post-ER VTCs but before their delivery to the Golgi stack. This defines a novel function for p115, and identifies the first step of membrane transport in which p115 participates.


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)

p115 depletion blocks 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 transport cocktails containing p115-depleted cytosol (A–C) or complete cytosol (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti–VSV-G protein (A and D) and anti–Mann II (B and E) antibodies. Depletion of p115 from the transport assay had no effect on VSV-G protein exit from the ER, but prevented VSV-G protein transport to the Golgi (A–C) and caused accumulation of VSV-G protein in peripheral VTCs lacking Mann II. In the presence of complete cytosol, 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 8: p115 depletion blocks 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 transport cocktails containing p115-depleted cytosol (A–C) or complete cytosol (D–F). After transport at 32°C for 90 min, cells were processed by double label immunofluorescence using anti–VSV-G protein (A and D) and anti–Mann II (B and E) antibodies. Depletion of p115 from the transport assay had no effect on VSV-G protein exit from the ER, but prevented VSV-G protein transport to the Golgi (A–C) and caused accumulation of VSV-G protein in peripheral VTCs lacking Mann II. In the presence of complete cytosol, VSV-G protein was efficiently delivered to the Golgi (D–F). Bar 10 μm.
Mentions: When p115-depleted cytosol (analogous to that unable to support the biochemical assay in Fig. 5 C, lane 4) was used in the morphological transport assay, VSV-G protein was also not transported to the Golgi, as evidenced by its lack of colocalization with Mann II (Fig. 8, A–C). Two representative cells from two different experiments are shown in these panels. VSV-G protein localized predominantly to peripheral VTCs, indistinguishable from those seen in the presence of anti–p115 antibodies (compare to Fig. 7 B). VSV-G protein was almost exclusively in peripheral VTCs, and was not present to any significant extent in the ER, confirming that p115 is not involved in ER exit and delivery of cargo to more distal transport intermediates. VSV-G protein was efficiently transported to the Golgi when complete cytosol was used (Fig. 8, D–F). Taken together, these results indicate that p115 function is required after the formation of post-ER VTCs but before their delivery to the Golgi stack. This defines a novel function for p115, and identifies the first step of membrane transport in which p115 participates.

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