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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 Golgi complex reassembly during BFA wash-out. WIF-B cells were treated with BFA for 30 min, and then injected with mAbs against p115 mixed with TR-dextran. Cells were fixed after 10 min (A and B), 30 min (C and D), or 120 min (E and F) of BFA wash-out, and processed for immunofluorescence using antibodies against Mann II (A, C, and E). Injected cells were identified by their content of TR-dextran (B, D, and F), and are traced in outline in A, C, and E. After 10 min of BFA wash-out, Mann II relocation to punctate structures is indistinguishable in injected and uninjected cells. After 30 and 120 min of BFA wash-out, Mann II appears in morphologically normal Golgi structures in uninjected cells, but remains in punctate structures in injected cells. Arrowheads mark Golgi in injected cells, arrows point to Golgi in uninjected cells. Asterisk denotes a more compact Golgi complex in injected cells.
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Figure 3: Antibodies against p115 block Golgi complex reassembly during BFA wash-out. WIF-B cells were treated with BFA for 30 min, and then injected with mAbs against p115 mixed with TR-dextran. Cells were fixed after 10 min (A and B), 30 min (C and D), or 120 min (E and F) of BFA wash-out, and processed for immunofluorescence using antibodies against Mann II (A, C, and E). Injected cells were identified by their content of TR-dextran (B, D, and F), and are traced in outline in A, C, and E. After 10 min of BFA wash-out, Mann II relocation to punctate structures is indistinguishable in injected and uninjected cells. After 30 and 120 min of BFA wash-out, Mann II appears in morphologically normal Golgi structures in uninjected cells, but remains in punctate structures in injected cells. Arrowheads mark Golgi in injected cells, arrows point to Golgi in uninjected cells. Asterisk denotes a more compact Golgi complex in injected cells.

Mentions: To examine if the antibodies inhibit the movement of Mann II from the ER, WIF-B cells were first treated with BFA for 30 min to relocate Mann II from the Golgi to the ER, and then injected with anti–p115 antibodies. The temporal sequence of Golgi recovery after BFA wash-out was followed. As shown in Fig. 3 A, punctate structures dispersed throughout the cell were detected after a 10-min BFA wash-out in both, uninjected and injected cells. The size and distribution of the elements were comparable, suggesting that the antibodies do not block early stages of BFA recovery. A difference was seen after 30 min of BFA wash-out, when defined Golgi structures could be seen in uninjected cells (C, arrows), while significantly smaller, dispersed structures were present in the injected cells (C, arrowheads). The effect was also evident after 120 min of BFA wash-out, when compact Golgi structures were seen in uninjected cells (E, arrows), whereas dispersed punctate structures persisted in the injected cells (E, arrowheads). In some injected cells, a more Golgi-like Mann II pattern was seen (E, asterisk), but the structure appeared less compact and organized. These results suggest that anti–p115 antibodies prevent the reassembly of normal Golgi complexes.


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 Golgi complex reassembly during BFA wash-out. WIF-B cells were treated with BFA for 30 min, and then injected with mAbs against p115 mixed with TR-dextran. Cells were fixed after 10 min (A and B), 30 min (C and D), or 120 min (E and F) of BFA wash-out, and processed for immunofluorescence using antibodies against Mann II (A, C, and E). Injected cells were identified by their content of TR-dextran (B, D, and F), and are traced in outline in A, C, and E. After 10 min of BFA wash-out, Mann II relocation to punctate structures is indistinguishable in injected and uninjected cells. After 30 and 120 min of BFA wash-out, Mann II appears in morphologically normal Golgi structures in uninjected cells, but remains in punctate structures in injected cells. Arrowheads mark Golgi in injected cells, arrows point to Golgi in uninjected cells. Asterisk denotes a more compact Golgi complex in injected cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168100&req=5

Figure 3: Antibodies against p115 block Golgi complex reassembly during BFA wash-out. WIF-B cells were treated with BFA for 30 min, and then injected with mAbs against p115 mixed with TR-dextran. Cells were fixed after 10 min (A and B), 30 min (C and D), or 120 min (E and F) of BFA wash-out, and processed for immunofluorescence using antibodies against Mann II (A, C, and E). Injected cells were identified by their content of TR-dextran (B, D, and F), and are traced in outline in A, C, and E. After 10 min of BFA wash-out, Mann II relocation to punctate structures is indistinguishable in injected and uninjected cells. After 30 and 120 min of BFA wash-out, Mann II appears in morphologically normal Golgi structures in uninjected cells, but remains in punctate structures in injected cells. Arrowheads mark Golgi in injected cells, arrows point to Golgi in uninjected cells. Asterisk denotes a more compact Golgi complex in injected cells.
Mentions: To examine if the antibodies inhibit the movement of Mann II from the ER, WIF-B cells were first treated with BFA for 30 min to relocate Mann II from the Golgi to the ER, and then injected with anti–p115 antibodies. The temporal sequence of Golgi recovery after BFA wash-out was followed. As shown in Fig. 3 A, punctate structures dispersed throughout the cell were detected after a 10-min BFA wash-out in both, uninjected and injected cells. The size and distribution of the elements were comparable, suggesting that the antibodies do not block early stages of BFA recovery. A difference was seen after 30 min of BFA wash-out, when defined Golgi structures could be seen in uninjected cells (C, arrows), while significantly smaller, dispersed structures were present in the injected cells (C, arrowheads). The effect was also evident after 120 min of BFA wash-out, when compact Golgi structures were seen in uninjected cells (E, arrows), whereas dispersed punctate structures persisted in the injected cells (E, arrowheads). In some injected cells, a more Golgi-like Mann II pattern was seen (E, asterisk), but the structure appeared less compact and organized. These results suggest that anti–p115 antibodies prevent the reassembly of normal Golgi complexes.

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