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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 is essential for ER to Golgi transport. ER to Golgi transport was performed in semi-intact NRK cells. Transport is measured as the percentage of VSV-G protein processed from the endo-H–sensitive (S) to the endo-H–resistant (R) form. (A) Transport reactions contained complete transport cocktail (lanes 2–6), or cocktail with ATP-depleting system (lane 1). Reactions were supplemented with increasing amounts of affinity-purified anti–p115 antibodies (lanes 3–6). Transport of VSV-G protein was proportionally inhibited in the presence of antibodies against p115. Analogous gels (n = 3) were quantitated by densitometry and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. (B) Transport reactions contained complete transport cocktail (lane 2), cocktail with ATP-depleting system (lane 1), or cocktail supplemented with anti–p115 antibodies preincubated with GST-p115 (lane 3), or GST (lane 4). Preincubation of anti–p115 antibodies with GST-p115 neutralized their inhibitory effect on transport. (C) Transport reactions contained complete transport cocktail (lanes 2–7), or cocktail with ATP-depleting system (lane 1). In lane 3, transport cocktail contained cytosol preincubated with control IgGs. In lane 4, transport cocktail contained cytosol preincubated with anti–p115 antibodies. Reduced level of p115 is visible in lane 4 and leads to inhibition of VSV-G protein transport. Increasing amounts of purified p115 were added to the cytosol shown in lane 4. Addition of purified p115 overcame the inhibitory effect of p115 removal and supported VSV-G protein transport (lanes 5–7). Analogous gels (n = 3) were quantitated by densitometry, and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The same amount of p115 was used in lane 1 as in lane 2 and only reaction in lane 2 was analyzed. (D) Rat liver cytosol was incubated with anti–p115 antibodies or control IgGs cross-linked to protein A–Sepharose. Bound material was eluted and analyzed by SDS-PAGE. An ∼110-kD band was visible after Coomassie blue staining in material eluted from anti-p115 column (lane 1) but not in control eluate (lane 2). The ∼110-kD band was excised, digested with trypsin, and the resulting peptides analyzed by MALDI mass spectrometry. The peptide mass map of the 10 most abundant peptides (marked by asterisks) matched the sequence of p115.
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Figure 5: p115 is essential for ER to Golgi transport. ER to Golgi transport was performed in semi-intact NRK cells. Transport is measured as the percentage of VSV-G protein processed from the endo-H–sensitive (S) to the endo-H–resistant (R) form. (A) Transport reactions contained complete transport cocktail (lanes 2–6), or cocktail with ATP-depleting system (lane 1). Reactions were supplemented with increasing amounts of affinity-purified anti–p115 antibodies (lanes 3–6). Transport of VSV-G protein was proportionally inhibited in the presence of antibodies against p115. Analogous gels (n = 3) were quantitated by densitometry and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. (B) Transport reactions contained complete transport cocktail (lane 2), cocktail with ATP-depleting system (lane 1), or cocktail supplemented with anti–p115 antibodies preincubated with GST-p115 (lane 3), or GST (lane 4). Preincubation of anti–p115 antibodies with GST-p115 neutralized their inhibitory effect on transport. (C) Transport reactions contained complete transport cocktail (lanes 2–7), or cocktail with ATP-depleting system (lane 1). In lane 3, transport cocktail contained cytosol preincubated with control IgGs. In lane 4, transport cocktail contained cytosol preincubated with anti–p115 antibodies. Reduced level of p115 is visible in lane 4 and leads to inhibition of VSV-G protein transport. Increasing amounts of purified p115 were added to the cytosol shown in lane 4. Addition of purified p115 overcame the inhibitory effect of p115 removal and supported VSV-G protein transport (lanes 5–7). Analogous gels (n = 3) were quantitated by densitometry, and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The same amount of p115 was used in lane 1 as in lane 2 and only reaction in lane 2 was analyzed. (D) Rat liver cytosol was incubated with anti–p115 antibodies or control IgGs cross-linked to protein A–Sepharose. Bound material was eluted and analyzed by SDS-PAGE. An ∼110-kD band was visible after Coomassie blue staining in material eluted from anti-p115 column (lane 1) but not in control eluate (lane 2). The ∼110-kD band was excised, digested with trypsin, and the resulting peptides analyzed by MALDI mass spectrometry. The peptide mass map of the 10 most abundant peptides (marked by asterisks) matched the sequence of p115.

Mentions: Although p115 has been identified as a cis- to medial-Golgi transport factor (Waters et al. 1992), its yeast homologue, Uso1p, has been shown to act in ER to Golgi traffic (Nakajima et al. 1991; Cao et al. 1998). To examine directly p115 participation in ER to Golgi traffic, we used a previously developed semi-intact cell transport assay (Beckers et al. 1987). NRK cells were infected with VSVtsO45 and radiolabeled at 42°C. Cells were permeabilized to remove endogenous cytosol, supplemented with exogenous transport cocktails, and shifted to 32°C to initiate VSV-G protein transport. Delivery of VSV-G protein to the Golgi was assessed by its carbohydrate processing, as defined by endo-H resistance. VSV-G protein oligosaccharide chains are processed during transport by the sequential actions of enzymes localized throughout the Golgi stack. The processing involves the sequential function of Mann I and N-acetylglucosamine transferase I (NAGT-1), both considered cis-Golgi enzymes (Schwaninger et al. 1992b), and of Mann II, localized in the medial/trans Golgi stack (Velasco et al. 1993). After processing by Mann I, VSV-G acquires endo-D sensitivity, whereas subsequent processing by NAGT-1 and Mann II confers endo-H resistance. As shown in Fig. 5 A, when complete transport cocktail was added to permeabilized cells, ∼50% of VSV-G protein was processed to an endo-H–resistant form (top band, lane 2), and this is set as 100% processing. The percent processing is analogous to the level of processing reported previously (Tisdale and Balch 1996; Subramanian et al., 1996). In contrast, when transport was analyzed with an ATP-depleting system, VSV-G protein remained sensitive to endo-H (bottom band, lane 1), and this is set as 0% processing. The addition of increasing amounts of affinity-purified anti–p115 antibodies (from 0.1 to 0.8 μg) led to a dose-dependent inhibition of VSV-G protein processing to the endo-H–resistant form (lanes 3–6). To provide quantitative data on VSV-G processing, analogous data from repeated experiments (n = 3) were evaluated by densitometry, and the average of relative percent is presented in the accompanying bar graph. The relative processing was reduced by 15% in the presence of 0.1 μg of antibody with >80% inhibition when 0.4 μg of anti–p115 antibodies were added. When preimmune antibodies were added to the transport assay, normal processing of VSV-G protein was observed (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)

p115 is essential for ER to Golgi transport. ER to Golgi transport was performed in semi-intact NRK cells. Transport is measured as the percentage of VSV-G protein processed from the endo-H–sensitive (S) to the endo-H–resistant (R) form. (A) Transport reactions contained complete transport cocktail (lanes 2–6), or cocktail with ATP-depleting system (lane 1). Reactions were supplemented with increasing amounts of affinity-purified anti–p115 antibodies (lanes 3–6). Transport of VSV-G protein was proportionally inhibited in the presence of antibodies against p115. Analogous gels (n = 3) were quantitated by densitometry and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. (B) Transport reactions contained complete transport cocktail (lane 2), cocktail with ATP-depleting system (lane 1), or cocktail supplemented with anti–p115 antibodies preincubated with GST-p115 (lane 3), or GST (lane 4). Preincubation of anti–p115 antibodies with GST-p115 neutralized their inhibitory effect on transport. (C) Transport reactions contained complete transport cocktail (lanes 2–7), or cocktail with ATP-depleting system (lane 1). In lane 3, transport cocktail contained cytosol preincubated with control IgGs. In lane 4, transport cocktail contained cytosol preincubated with anti–p115 antibodies. Reduced level of p115 is visible in lane 4 and leads to inhibition of VSV-G protein transport. Increasing amounts of purified p115 were added to the cytosol shown in lane 4. Addition of purified p115 overcame the inhibitory effect of p115 removal and supported VSV-G protein transport (lanes 5–7). Analogous gels (n = 3) were quantitated by densitometry, and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The same amount of p115 was used in lane 1 as in lane 2 and only reaction in lane 2 was analyzed. (D) Rat liver cytosol was incubated with anti–p115 antibodies or control IgGs cross-linked to protein A–Sepharose. Bound material was eluted and analyzed by SDS-PAGE. An ∼110-kD band was visible after Coomassie blue staining in material eluted from anti-p115 column (lane 1) but not in control eluate (lane 2). The ∼110-kD band was excised, digested with trypsin, and the resulting peptides analyzed by MALDI mass spectrometry. The peptide mass map of the 10 most abundant peptides (marked by asterisks) matched the sequence of p115.
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Related In: Results  -  Collection

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Figure 5: p115 is essential for ER to Golgi transport. ER to Golgi transport was performed in semi-intact NRK cells. Transport is measured as the percentage of VSV-G protein processed from the endo-H–sensitive (S) to the endo-H–resistant (R) form. (A) Transport reactions contained complete transport cocktail (lanes 2–6), or cocktail with ATP-depleting system (lane 1). Reactions were supplemented with increasing amounts of affinity-purified anti–p115 antibodies (lanes 3–6). Transport of VSV-G protein was proportionally inhibited in the presence of antibodies against p115. Analogous gels (n = 3) were quantitated by densitometry and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. (B) Transport reactions contained complete transport cocktail (lane 2), cocktail with ATP-depleting system (lane 1), or cocktail supplemented with anti–p115 antibodies preincubated with GST-p115 (lane 3), or GST (lane 4). Preincubation of anti–p115 antibodies with GST-p115 neutralized their inhibitory effect on transport. (C) Transport reactions contained complete transport cocktail (lanes 2–7), or cocktail with ATP-depleting system (lane 1). In lane 3, transport cocktail contained cytosol preincubated with control IgGs. In lane 4, transport cocktail contained cytosol preincubated with anti–p115 antibodies. Reduced level of p115 is visible in lane 4 and leads to inhibition of VSV-G protein transport. Increasing amounts of purified p115 were added to the cytosol shown in lane 4. Addition of purified p115 overcame the inhibitory effect of p115 removal and supported VSV-G protein transport (lanes 5–7). Analogous gels (n = 3) were quantitated by densitometry, and the averages are presented in the bar graph. Transport in lane 1 is set as 0% and in lane 2 as 100%. An aliquot of each transport reaction was probed by immunoblotting with anti–p115 antibodies and the immunoblot is shown in panel p115. The same amount of p115 was used in lane 1 as in lane 2 and only reaction in lane 2 was analyzed. (D) Rat liver cytosol was incubated with anti–p115 antibodies or control IgGs cross-linked to protein A–Sepharose. Bound material was eluted and analyzed by SDS-PAGE. An ∼110-kD band was visible after Coomassie blue staining in material eluted from anti-p115 column (lane 1) but not in control eluate (lane 2). The ∼110-kD band was excised, digested with trypsin, and the resulting peptides analyzed by MALDI mass spectrometry. The peptide mass map of the 10 most abundant peptides (marked by asterisks) matched the sequence of p115.
Mentions: Although p115 has been identified as a cis- to medial-Golgi transport factor (Waters et al. 1992), its yeast homologue, Uso1p, has been shown to act in ER to Golgi traffic (Nakajima et al. 1991; Cao et al. 1998). To examine directly p115 participation in ER to Golgi traffic, we used a previously developed semi-intact cell transport assay (Beckers et al. 1987). NRK cells were infected with VSVtsO45 and radiolabeled at 42°C. Cells were permeabilized to remove endogenous cytosol, supplemented with exogenous transport cocktails, and shifted to 32°C to initiate VSV-G protein transport. Delivery of VSV-G protein to the Golgi was assessed by its carbohydrate processing, as defined by endo-H resistance. VSV-G protein oligosaccharide chains are processed during transport by the sequential actions of enzymes localized throughout the Golgi stack. The processing involves the sequential function of Mann I and N-acetylglucosamine transferase I (NAGT-1), both considered cis-Golgi enzymes (Schwaninger et al. 1992b), and of Mann II, localized in the medial/trans Golgi stack (Velasco et al. 1993). After processing by Mann I, VSV-G acquires endo-D sensitivity, whereas subsequent processing by NAGT-1 and Mann II confers endo-H resistance. As shown in Fig. 5 A, when complete transport cocktail was added to permeabilized cells, ∼50% of VSV-G protein was processed to an endo-H–resistant form (top band, lane 2), and this is set as 100% processing. The percent processing is analogous to the level of processing reported previously (Tisdale and Balch 1996; Subramanian et al., 1996). In contrast, when transport was analyzed with an ATP-depleting system, VSV-G protein remained sensitive to endo-H (bottom band, lane 1), and this is set as 0% processing. The addition of increasing amounts of affinity-purified anti–p115 antibodies (from 0.1 to 0.8 μg) led to a dose-dependent inhibition of VSV-G protein processing to the endo-H–resistant form (lanes 3–6). To provide quantitative data on VSV-G processing, analogous data from repeated experiments (n = 3) were evaluated by densitometry, and the average of relative percent is presented in the accompanying bar graph. The relative processing was reduced by 15% in the presence of 0.1 μg of antibody with >80% inhibition when 0.4 μg of anti–p115 antibodies were added. When preimmune antibodies were added to the transport assay, normal processing of VSV-G protein was observed (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