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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 do not block BFA-induced retrograde Golgi to ER traffic. Mouse mAbs against p115 were microinjected into the cytoplasm of WIF-B cells. Cells were fixed immediately after injection (A and B) or after a 30-min BFA treatment (C and D). Cell were processed for double label immunofluorescence using antibodies against Mann II (A and C) or against mouse IgG (B and D). Relocation of Mann II from the Golgi to the ER appears indistinguishable in injected and uninjected cells. Golgi remnants were detected in some cells after the 30-min BFA treatment (C, arrowheads).
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Figure 2: Antibodies against p115 do not block BFA-induced retrograde Golgi to ER traffic. Mouse mAbs against p115 were microinjected into the cytoplasm of WIF-B cells. Cells were fixed immediately after injection (A and B) or after a 30-min BFA treatment (C and D). Cell were processed for double label immunofluorescence using antibodies against Mann II (A and C) or against mouse IgG (B and D). Relocation of Mann II from the Golgi to the ER appears indistinguishable in injected and uninjected cells. Golgi remnants were detected in some cells after the 30-min BFA treatment (C, arrowheads).

Mentions: The redistribution of Mann II from the Golgi into the ER in control WIF-B cells or in cells injected with anti–p115 antibodies was examined first. As shown in Fig. 2, Mann II was detected in a normal perinuclear Golgi pattern in uninjected cells or in injected cells immediately after the antibody injection and before BFA treatment (A). The injected cells were identified by the presence of anti–p115 IgG (B). When an analogous field of cells was treated with BFA for 30 min, Mann II relocated from the Golgi to the ER in uninjected and injected cells (C). Only a single time point was analyzed, and it remains possible that the kinetics of Mann II redistribution may have varied in injected and uninjected cells. Some Golgi staining was still evident in uninjected and injected cells after the BFA treatment (C, arrowheads). After 30 min, the majority of anti–p115 IgG was associated with membranes, presumably representing p115 localization (D), as shown previously (Nelson et al. 1998). The results suggest that anti–p115 antibodies do not block the Golgi to ER redistribution of Mann II induced by BFA treatment.


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 do not block BFA-induced retrograde Golgi to ER traffic. Mouse mAbs against p115 were microinjected into the cytoplasm of WIF-B cells. Cells were fixed immediately after injection (A and B) or after a 30-min BFA treatment (C and D). Cell were processed for double label immunofluorescence using antibodies against Mann II (A and C) or against mouse IgG (B and D). Relocation of Mann II from the Golgi to the ER appears indistinguishable in injected and uninjected cells. Golgi remnants were detected in some cells after the 30-min BFA treatment (C, arrowheads).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Antibodies against p115 do not block BFA-induced retrograde Golgi to ER traffic. Mouse mAbs against p115 were microinjected into the cytoplasm of WIF-B cells. Cells were fixed immediately after injection (A and B) or after a 30-min BFA treatment (C and D). Cell were processed for double label immunofluorescence using antibodies against Mann II (A and C) or against mouse IgG (B and D). Relocation of Mann II from the Golgi to the ER appears indistinguishable in injected and uninjected cells. Golgi remnants were detected in some cells after the 30-min BFA treatment (C, arrowheads).
Mentions: The redistribution of Mann II from the Golgi into the ER in control WIF-B cells or in cells injected with anti–p115 antibodies was examined first. As shown in Fig. 2, Mann II was detected in a normal perinuclear Golgi pattern in uninjected cells or in injected cells immediately after the antibody injection and before BFA treatment (A). The injected cells were identified by the presence of anti–p115 IgG (B). When an analogous field of cells was treated with BFA for 30 min, Mann II relocated from the Golgi to the ER in uninjected and injected cells (C). Only a single time point was analyzed, and it remains possible that the kinetics of Mann II redistribution may have varied in injected and uninjected cells. Some Golgi staining was still evident in uninjected and injected cells after the BFA treatment (C, arrowheads). After 30 min, the majority of anti–p115 IgG was associated with membranes, presumably representing p115 localization (D), as shown previously (Nelson et al. 1998). The results suggest that anti–p115 antibodies do not block the Golgi to ER redistribution of Mann II induced by BFA treatment.

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