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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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VSV-G protein is differentially endo-D resistant/sensitive in LEC-1 and NRK cells. (A and B) ER to Golgi transport was performed in semi-intact LEC-1 cells. After transport, the sensitivity/resistance of VSV-G protein to endo-D was analyzed. (A) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (−ATP), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (+ATP), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing control antibodies (control) or anti–p115 antibodies (anti-p115) are used. (B) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (lane 1), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (lane 2), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing cytosol immunodepleted with control antibodies (lane 3), preimmune antibodies (lane 4), or anti–p115 antibodies (lanes 5 and 6) are used. Analogous gels (n = 3) were quantitated by densitometry and averages are presented as a bar graph. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The relative amounts of p115 in each transport reaction are presented in the bar graph. The amount of p115 in lane 1 is set as 100%. (C) LEC-1 cells were infected with VSVtsO45 for 3 h at 42°C, and either analyzed directly or incubated at 32°C for additional 1 h or at 15°C for additional 3 h before analysis. Cells were collected and analyzed directly or after endo-D digestion. Processing of VSV-G protein from the endo-D–resistant (R) to the endo-D–sensitive (S) form is shown. A single VSV-G band is seen in untreated samples regardless of incubation temperature (lanes 4–6). VSV-G is resistant to endo-D when retained in the ER during the 42°C incubation (lane 1), but becomes endo-D sensitive when transported to the Golgi during a 32°C incubation (lane 2) or when arrested in peripheral VTCs during a 15°C incubation (lane 3). (D, lanes 1–3 and 5–7) ER to Golgi transport was performed in semi-intact NRK cells. After transport, the sensitivity/resistance of VSV-G protein to endo-H and endo-D was analyzed in parallel. VSV-G protein is endo-H sensitive (lane 1) and endo-D resistant (lane 5) when transport cocktail lacking ATP is used. VSV-G protein is endo-H resistant (lane 2) and endo-D resistant (lane 6) when complete transport cocktail is used. VSV-G protein is endo-H sensitive (lane 3) and endo-D resistant (lane 7) when complete transport cocktails containing anti–p115 antibodies are used. (D, lanes 4 and 8) NRK cells were infected with VSVtsO45 (3 h at 32°C), pulsed-labeled at the restrictive temperature (10 min at 42°C) and chased in complete medium for 3 h at 15°C. Cells were collected and digested with either endo-H (lane 4) or endo-D (lane 8). VSV-G protein is endo-H sensitive and endo-D resistant when arrested in peripheral VTCs at 15°C.
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Figure 9: VSV-G protein is differentially endo-D resistant/sensitive in LEC-1 and NRK cells. (A and B) ER to Golgi transport was performed in semi-intact LEC-1 cells. After transport, the sensitivity/resistance of VSV-G protein to endo-D was analyzed. (A) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (−ATP), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (+ATP), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing control antibodies (control) or anti–p115 antibodies (anti-p115) are used. (B) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (lane 1), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (lane 2), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing cytosol immunodepleted with control antibodies (lane 3), preimmune antibodies (lane 4), or anti–p115 antibodies (lanes 5 and 6) are used. Analogous gels (n = 3) were quantitated by densitometry and averages are presented as a bar graph. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The relative amounts of p115 in each transport reaction are presented in the bar graph. The amount of p115 in lane 1 is set as 100%. (C) LEC-1 cells were infected with VSVtsO45 for 3 h at 42°C, and either analyzed directly or incubated at 32°C for additional 1 h or at 15°C for additional 3 h before analysis. Cells were collected and analyzed directly or after endo-D digestion. Processing of VSV-G protein from the endo-D–resistant (R) to the endo-D–sensitive (S) form is shown. A single VSV-G band is seen in untreated samples regardless of incubation temperature (lanes 4–6). VSV-G is resistant to endo-D when retained in the ER during the 42°C incubation (lane 1), but becomes endo-D sensitive when transported to the Golgi during a 32°C incubation (lane 2) or when arrested in peripheral VTCs during a 15°C incubation (lane 3). (D, lanes 1–3 and 5–7) ER to Golgi transport was performed in semi-intact NRK cells. After transport, the sensitivity/resistance of VSV-G protein to endo-H and endo-D was analyzed in parallel. VSV-G protein is endo-H sensitive (lane 1) and endo-D resistant (lane 5) when transport cocktail lacking ATP is used. VSV-G protein is endo-H resistant (lane 2) and endo-D resistant (lane 6) when complete transport cocktail is used. VSV-G protein is endo-H sensitive (lane 3) and endo-D resistant (lane 7) when complete transport cocktails containing anti–p115 antibodies are used. (D, lanes 4 and 8) NRK cells were infected with VSVtsO45 (3 h at 32°C), pulsed-labeled at the restrictive temperature (10 min at 42°C) and chased in complete medium for 3 h at 15°C. Cells were collected and digested with either endo-H (lane 4) or endo-D (lane 8). VSV-G protein is endo-H sensitive and endo-D resistant when arrested in peripheral VTCs at 15°C.

Mentions: Mutant LEC-1 cells that lack NAGT-1 were used first because in these cells oligosaccharide modifications stop after Mann I processing (Stanley et al. 1975). VSV-G protein is endo-D resistant while in the ER and becomes endo-D sensitive after being transported and processed by Mann I. When infected cells were permeabilized and used in the semi-intact cell transport assay in the presence of an ATP-depleting system, VSV-G protein was endo-D resistant (Fig. 9 A, −ATP). When complete transport cocktail was used, a proportion (∼60%) of VSV-G protein became endo-D sensitive, and this is taken as the standard to which other reactions are compared (Fig. 9 A, +ATP). Unexpectedly, VSV-G protein was processed to the endo-D–sensitive form when anti–p115 antibodies were added to the transport assay (Fig. 9 A, anti-p115) in an amount analogous to that found to block acquisition of endo-H resistance in NRK cells (Fig. 5 A, lane 6). Addition of control antibodies had no effect on VSV-G protein processing (Fig. 9 A, control). Similarly, when p115-depleted cytosol was used in the transport assay, VSV-G protein was processed to the endo-D–sensitive form (Fig. 9 B, gel and bar graph, lanes 5 and 6). Even a significant depletion of p115 (>80% of p115 was depleted in lanes 5 and 6 as compared with lane 2) had no effect on the amount of VSV-G protein sensitive to endo-D. The level of p115 (∼20% of control) that supported the processing to the endo-D–sensitive form in LEC-1 cells was unable to support processing to the endo-H–resistant form in NRK cells (compare Fig. 9 B, lane 6, to Fig. 5 C, lane 4). The level of endo-D processing in reactions containing cytosol immunodepleted with anti–p115 antibodies was similar to that when control (Fig. 9 B, lane 3) or preimmune (Fig. 9 B, lane 4) antibodies were used for immunodepletion.


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)

VSV-G protein is differentially endo-D resistant/sensitive in LEC-1 and NRK cells. (A and B) ER to Golgi transport was performed in semi-intact LEC-1 cells. After transport, the sensitivity/resistance of VSV-G protein to endo-D was analyzed. (A) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (−ATP), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (+ATP), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing control antibodies (control) or anti–p115 antibodies (anti-p115) are used. (B) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (lane 1), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (lane 2), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing cytosol immunodepleted with control antibodies (lane 3), preimmune antibodies (lane 4), or anti–p115 antibodies (lanes 5 and 6) are used. Analogous gels (n = 3) were quantitated by densitometry and averages are presented as a bar graph. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The relative amounts of p115 in each transport reaction are presented in the bar graph. The amount of p115 in lane 1 is set as 100%. (C) LEC-1 cells were infected with VSVtsO45 for 3 h at 42°C, and either analyzed directly or incubated at 32°C for additional 1 h or at 15°C for additional 3 h before analysis. Cells were collected and analyzed directly or after endo-D digestion. Processing of VSV-G protein from the endo-D–resistant (R) to the endo-D–sensitive (S) form is shown. A single VSV-G band is seen in untreated samples regardless of incubation temperature (lanes 4–6). VSV-G is resistant to endo-D when retained in the ER during the 42°C incubation (lane 1), but becomes endo-D sensitive when transported to the Golgi during a 32°C incubation (lane 2) or when arrested in peripheral VTCs during a 15°C incubation (lane 3). (D, lanes 1–3 and 5–7) ER to Golgi transport was performed in semi-intact NRK cells. After transport, the sensitivity/resistance of VSV-G protein to endo-H and endo-D was analyzed in parallel. VSV-G protein is endo-H sensitive (lane 1) and endo-D resistant (lane 5) when transport cocktail lacking ATP is used. VSV-G protein is endo-H resistant (lane 2) and endo-D resistant (lane 6) when complete transport cocktail is used. VSV-G protein is endo-H sensitive (lane 3) and endo-D resistant (lane 7) when complete transport cocktails containing anti–p115 antibodies are used. (D, lanes 4 and 8) NRK cells were infected with VSVtsO45 (3 h at 32°C), pulsed-labeled at the restrictive temperature (10 min at 42°C) and chased in complete medium for 3 h at 15°C. Cells were collected and digested with either endo-H (lane 4) or endo-D (lane 8). VSV-G protein is endo-H sensitive and endo-D resistant when arrested in peripheral VTCs at 15°C.
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Figure 9: VSV-G protein is differentially endo-D resistant/sensitive in LEC-1 and NRK cells. (A and B) ER to Golgi transport was performed in semi-intact LEC-1 cells. After transport, the sensitivity/resistance of VSV-G protein to endo-D was analyzed. (A) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (−ATP), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (+ATP), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing control antibodies (control) or anti–p115 antibodies (anti-p115) are used. (B) VSV-G protein is endo-D resistant when transport cocktail lacking ATP is used (lane 1), and this is set as 0% relative processing. A proportion of VSV-G protein is endo-D sensitive when complete transport cocktail is used (lane 2), and this is set as 100% relative processing. A similar proportion of VSV-G protein is endo-D sensitive when complete transport cocktails containing cytosol immunodepleted with control antibodies (lane 3), preimmune antibodies (lane 4), or anti–p115 antibodies (lanes 5 and 6) are used. Analogous gels (n = 3) were quantitated by densitometry and averages are presented as a bar graph. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The relative amounts of p115 in each transport reaction are presented in the bar graph. The amount of p115 in lane 1 is set as 100%. (C) LEC-1 cells were infected with VSVtsO45 for 3 h at 42°C, and either analyzed directly or incubated at 32°C for additional 1 h or at 15°C for additional 3 h before analysis. Cells were collected and analyzed directly or after endo-D digestion. Processing of VSV-G protein from the endo-D–resistant (R) to the endo-D–sensitive (S) form is shown. A single VSV-G band is seen in untreated samples regardless of incubation temperature (lanes 4–6). VSV-G is resistant to endo-D when retained in the ER during the 42°C incubation (lane 1), but becomes endo-D sensitive when transported to the Golgi during a 32°C incubation (lane 2) or when arrested in peripheral VTCs during a 15°C incubation (lane 3). (D, lanes 1–3 and 5–7) ER to Golgi transport was performed in semi-intact NRK cells. After transport, the sensitivity/resistance of VSV-G protein to endo-H and endo-D was analyzed in parallel. VSV-G protein is endo-H sensitive (lane 1) and endo-D resistant (lane 5) when transport cocktail lacking ATP is used. VSV-G protein is endo-H resistant (lane 2) and endo-D resistant (lane 6) when complete transport cocktail is used. VSV-G protein is endo-H sensitive (lane 3) and endo-D resistant (lane 7) when complete transport cocktails containing anti–p115 antibodies are used. (D, lanes 4 and 8) NRK cells were infected with VSVtsO45 (3 h at 32°C), pulsed-labeled at the restrictive temperature (10 min at 42°C) and chased in complete medium for 3 h at 15°C. Cells were collected and digested with either endo-H (lane 4) or endo-D (lane 8). VSV-G protein is endo-H sensitive and endo-D resistant when arrested in peripheral VTCs at 15°C.
Mentions: Mutant LEC-1 cells that lack NAGT-1 were used first because in these cells oligosaccharide modifications stop after Mann I processing (Stanley et al. 1975). VSV-G protein is endo-D resistant while in the ER and becomes endo-D sensitive after being transported and processed by Mann I. When infected cells were permeabilized and used in the semi-intact cell transport assay in the presence of an ATP-depleting system, VSV-G protein was endo-D resistant (Fig. 9 A, −ATP). When complete transport cocktail was used, a proportion (∼60%) of VSV-G protein became endo-D sensitive, and this is taken as the standard to which other reactions are compared (Fig. 9 A, +ATP). Unexpectedly, VSV-G protein was processed to the endo-D–sensitive form when anti–p115 antibodies were added to the transport assay (Fig. 9 A, anti-p115) in an amount analogous to that found to block acquisition of endo-H resistance in NRK cells (Fig. 5 A, lane 6). Addition of control antibodies had no effect on VSV-G protein processing (Fig. 9 A, control). Similarly, when p115-depleted cytosol was used in the transport assay, VSV-G protein was processed to the endo-D–sensitive form (Fig. 9 B, gel and bar graph, lanes 5 and 6). Even a significant depletion of p115 (>80% of p115 was depleted in lanes 5 and 6 as compared with lane 2) had no effect on the amount of VSV-G protein sensitive to endo-D. The level of p115 (∼20% of control) that supported the processing to the endo-D–sensitive form in LEC-1 cells was unable to support processing to the endo-H–resistant form in NRK cells (compare Fig. 9 B, lane 6, to Fig. 5 C, lane 4). The level of endo-D processing in reactions containing cytosol immunodepleted with anti–p115 antibodies was similar to that when control (Fig. 9 B, lane 3) or preimmune (Fig. 9 B, lane 4) antibodies were used for immunodepletion.

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