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p53/58 binds COPI and is required for selective transport through the early secretory pathway.

Tisdale EJ, Plutner H, Matteson J, Balch WE - J. Cell Biol. (1997)

Bottom Line: p53/58 is a transmembrane protein that continuously recycles between the ER and pre-Golgi intermediates composed of vesicular-tubular clusters (VTCs) found in the cell periphery and at the cis face of the Golgi complex.Consistent with a role for the KKXX retrieval motif found at the cytoplasmic carboxyl terminus of p53/58 in retrograde traffic, inhibition of transport through VTCs correlates with the ability of the antibody to block recruitment of COPI coats to the p53/58 cytoplasmic tail and to p53/58-containing membranes.We suggest that p53/58 function may be required for the coupled exchange of COPII for COPI coats during segregation of anterograde and retrograde transported proteins.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Department of Cell Biology, La Jolla, California 92037, USA.

ABSTRACT
p53/58 is a transmembrane protein that continuously recycles between the ER and pre-Golgi intermediates composed of vesicular-tubular clusters (VTCs) found in the cell periphery and at the cis face of the Golgi complex. We have generated an antibody that uniquely recognizes the p53/58 cytoplasmic tail. Here we present evidence that this antibody arrests the anterograde transport of vesicular stomatitis virus glycoprotein and leads to the accumulation of p58 in pre-Golgi intermediates. Consistent with a role for the KKXX retrieval motif found at the cytoplasmic carboxyl terminus of p53/58 in retrograde traffic, inhibition of transport through VTCs correlates with the ability of the antibody to block recruitment of COPI coats to the p53/58 cytoplasmic tail and to p53/58-containing membranes. We suggest that p53/58 function may be required for the coupled exchange of COPII for COPI coats during segregation of anterograde and retrograde transported proteins.

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The antitail antibody blocks transport before the delivery of cargo to the Golgi compartment containing α-1,2 mannosidase I. Semiintact CHO clone 15B cells were incubated at 32°C in  a complete transport cocktail for indicated time in the absence  (closed circles) or presence (open circles) of antitail antibody. The  amount of VSV-G processed to endo D–sensitive forms was determined as described in Materials and Methods. (Inset) Antibody inhibition precedes the Ca2+-dependent fusion of VTCs to  the Golgi stack. Semiintact CHO clone 15B cells were incubated  in a transport cocktail that contained 5 mM EGTA for 60 min at  32°C to accumulate VSV-G in post-ER, pre-Golgi VTCs (Pind et al.,  1994). The cells were pelleted, resuspended in a transport cocktail that contained either 5 mM EGTA (a), 0.1 μM Ca2+ (b), or  0.1 μM Ca2+ and 10 μg of antitail antibody (c), and incubated at  32°C for 90 min. The fraction of VSV-G processed to the endo  D–sensitive form was determined as described in Materials and  Methods.
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Figure 4: The antitail antibody blocks transport before the delivery of cargo to the Golgi compartment containing α-1,2 mannosidase I. Semiintact CHO clone 15B cells were incubated at 32°C in a complete transport cocktail for indicated time in the absence (closed circles) or presence (open circles) of antitail antibody. The amount of VSV-G processed to endo D–sensitive forms was determined as described in Materials and Methods. (Inset) Antibody inhibition precedes the Ca2+-dependent fusion of VTCs to the Golgi stack. Semiintact CHO clone 15B cells were incubated in a transport cocktail that contained 5 mM EGTA for 60 min at 32°C to accumulate VSV-G in post-ER, pre-Golgi VTCs (Pind et al., 1994). The cells were pelleted, resuspended in a transport cocktail that contained either 5 mM EGTA (a), 0.1 μM Ca2+ (b), or 0.1 μM Ca2+ and 10 μg of antitail antibody (c), and incubated at 32°C for 90 min. The fraction of VSV-G processed to the endo D–sensitive form was determined as described in Materials and Methods.

Mentions: To localize the step in transport sensitive to antibody, we made use of the CHO clone 15B cell line, which lacks the cis/medial-Golgi enzyme N-acetylglucosamine-transferase I (GlcNAC Tr I) (Tabas and Kornfeld, 1979). In 15B cells, processing of VSV-G oligosaccharides does not proceed beyond the Man5 form, which appears in response to trimming by α-mannosidase I found in the cis region of Golgi stack (Beckers et al., 1987). The Man5 form of VSV-G is uniquely susceptible to digestion with endo D (Beckers et al., 1987), providing a direct measure for VSV-G delivery to the cis-most face of the Golgi. As shown in Fig. 4 (closed circles), semiintact CHO 15B cells incubated in the absence of antibody at 32°C processed VSV-G to the Man5 form. In contrast, in the presence of antibody, processing was completely inhibited (Fig. 4, open circles).


p53/58 binds COPI and is required for selective transport through the early secretory pathway.

Tisdale EJ, Plutner H, Matteson J, Balch WE - J. Cell Biol. (1997)

The antitail antibody blocks transport before the delivery of cargo to the Golgi compartment containing α-1,2 mannosidase I. Semiintact CHO clone 15B cells were incubated at 32°C in  a complete transport cocktail for indicated time in the absence  (closed circles) or presence (open circles) of antitail antibody. The  amount of VSV-G processed to endo D–sensitive forms was determined as described in Materials and Methods. (Inset) Antibody inhibition precedes the Ca2+-dependent fusion of VTCs to  the Golgi stack. Semiintact CHO clone 15B cells were incubated  in a transport cocktail that contained 5 mM EGTA for 60 min at  32°C to accumulate VSV-G in post-ER, pre-Golgi VTCs (Pind et al.,  1994). The cells were pelleted, resuspended in a transport cocktail that contained either 5 mM EGTA (a), 0.1 μM Ca2+ (b), or  0.1 μM Ca2+ and 10 μg of antitail antibody (c), and incubated at  32°C for 90 min. The fraction of VSV-G processed to the endo  D–sensitive form was determined as described in Materials and  Methods.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139878&req=5

Figure 4: The antitail antibody blocks transport before the delivery of cargo to the Golgi compartment containing α-1,2 mannosidase I. Semiintact CHO clone 15B cells were incubated at 32°C in a complete transport cocktail for indicated time in the absence (closed circles) or presence (open circles) of antitail antibody. The amount of VSV-G processed to endo D–sensitive forms was determined as described in Materials and Methods. (Inset) Antibody inhibition precedes the Ca2+-dependent fusion of VTCs to the Golgi stack. Semiintact CHO clone 15B cells were incubated in a transport cocktail that contained 5 mM EGTA for 60 min at 32°C to accumulate VSV-G in post-ER, pre-Golgi VTCs (Pind et al., 1994). The cells were pelleted, resuspended in a transport cocktail that contained either 5 mM EGTA (a), 0.1 μM Ca2+ (b), or 0.1 μM Ca2+ and 10 μg of antitail antibody (c), and incubated at 32°C for 90 min. The fraction of VSV-G processed to the endo D–sensitive form was determined as described in Materials and Methods.
Mentions: To localize the step in transport sensitive to antibody, we made use of the CHO clone 15B cell line, which lacks the cis/medial-Golgi enzyme N-acetylglucosamine-transferase I (GlcNAC Tr I) (Tabas and Kornfeld, 1979). In 15B cells, processing of VSV-G oligosaccharides does not proceed beyond the Man5 form, which appears in response to trimming by α-mannosidase I found in the cis region of Golgi stack (Beckers et al., 1987). The Man5 form of VSV-G is uniquely susceptible to digestion with endo D (Beckers et al., 1987), providing a direct measure for VSV-G delivery to the cis-most face of the Golgi. As shown in Fig. 4 (closed circles), semiintact CHO 15B cells incubated in the absence of antibody at 32°C processed VSV-G to the Man5 form. In contrast, in the presence of antibody, processing was completely inhibited (Fig. 4, open circles).

Bottom Line: p53/58 is a transmembrane protein that continuously recycles between the ER and pre-Golgi intermediates composed of vesicular-tubular clusters (VTCs) found in the cell periphery and at the cis face of the Golgi complex.Consistent with a role for the KKXX retrieval motif found at the cytoplasmic carboxyl terminus of p53/58 in retrograde traffic, inhibition of transport through VTCs correlates with the ability of the antibody to block recruitment of COPI coats to the p53/58 cytoplasmic tail and to p53/58-containing membranes.We suggest that p53/58 function may be required for the coupled exchange of COPII for COPI coats during segregation of anterograde and retrograde transported proteins.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, Department of Cell Biology, La Jolla, California 92037, USA.

ABSTRACT
p53/58 is a transmembrane protein that continuously recycles between the ER and pre-Golgi intermediates composed of vesicular-tubular clusters (VTCs) found in the cell periphery and at the cis face of the Golgi complex. We have generated an antibody that uniquely recognizes the p53/58 cytoplasmic tail. Here we present evidence that this antibody arrests the anterograde transport of vesicular stomatitis virus glycoprotein and leads to the accumulation of p58 in pre-Golgi intermediates. Consistent with a role for the KKXX retrieval motif found at the cytoplasmic carboxyl terminus of p53/58 in retrograde traffic, inhibition of transport through VTCs correlates with the ability of the antibody to block recruitment of COPI coats to the p53/58 cytoplasmic tail and to p53/58-containing membranes. We suggest that p53/58 function may be required for the coupled exchange of COPII for COPI coats during segregation of anterograde and retrograde transported proteins.

Show MeSH
Related in: MedlinePlus