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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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Antibody to the p53/58 carboxyl tail inhibits transport  from the ER to the Golgi complex. (A) Semiintact NRK cells  were prepared and incubated in vitro as described in Materials  and Methods. Semiintact cells were incubated with the indicated  concentration of antibody (closed squares) or Fab fragments (open  squares) for 45 min on ice and then transferred to 32°C for 90  min. (Inset) Preabsorption of the antibody to the cytoplasmic tail  neutralizes its inhibitory property. Semiintact cells were incubated in the absence (a) or presence of 5 μg of antipeptide antibody (b–d) and pretreated as follows: (b) no pretreatment; (c)  antibody preincubated with a GST–p53/58 tail fusion protein  bound to glutathione Sepharose 4B beads; (d) antibody preincubated with GST-glutathione Sepharose 4B beads as described in  Materials and Methods. In c and d, the unbound fraction was  added to the transport assay.
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Figure 3: Antibody to the p53/58 carboxyl tail inhibits transport from the ER to the Golgi complex. (A) Semiintact NRK cells were prepared and incubated in vitro as described in Materials and Methods. Semiintact cells were incubated with the indicated concentration of antibody (closed squares) or Fab fragments (open squares) for 45 min on ice and then transferred to 32°C for 90 min. (Inset) Preabsorption of the antibody to the cytoplasmic tail neutralizes its inhibitory property. Semiintact cells were incubated in the absence (a) or presence of 5 μg of antipeptide antibody (b–d) and pretreated as follows: (b) no pretreatment; (c) antibody preincubated with a GST–p53/58 tail fusion protein bound to glutathione Sepharose 4B beads; (d) antibody preincubated with GST-glutathione Sepharose 4B beads as described in Materials and Methods. In c and d, the unbound fraction was added to the transport assay.

Mentions: Preincubation of semiintact NRK cells with affinitypurified antibody led to a dose-dependent inhibition of ER to Golgi transport (Fig. 3). The processing of VSV-G to endo H–resistant forms was reduced by 50% in the presence of ∼2 μg antibody with complete inhibition at 8–10 μg (Fig. 3, closed squares). This inhibition was not a consequence of protein aggregation as Fab fragments also inhibited acquisition of endo H resistance at a level comparable to that of the intact antibody (Fig. 3, open squares). To provide further proof that the block in transport was due to the specific neutralization of a p53/58 carboxyl-terminal epitope, antibody was incubated with a GST–p53/58 carboxyl tail fusion protein (GST–p53/58 tail) bound to glutathione Sepharose 4B. The resulting nonabsorbed fraction was tested for activity in the semiintact cell assay. Preincubation of the antibody with the GST–p53/58 tail beads efficiently neutralized its inhibitory effect on protein traffic (Fig. 3, inset, c). In contrast, inhibition was not affected by incubation of antibody with control GST beads that lacked the p53/58 tail (Fig. 3, inset, d). These results show that the antibody blocks transport in NRK cells through a specific interaction with p58 and is the first demonstration that p58 may be required for ER to Golgi transport.


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)

Antibody to the p53/58 carboxyl tail inhibits transport  from the ER to the Golgi complex. (A) Semiintact NRK cells  were prepared and incubated in vitro as described in Materials  and Methods. Semiintact cells were incubated with the indicated  concentration of antibody (closed squares) or Fab fragments (open  squares) for 45 min on ice and then transferred to 32°C for 90  min. (Inset) Preabsorption of the antibody to the cytoplasmic tail  neutralizes its inhibitory property. Semiintact cells were incubated in the absence (a) or presence of 5 μg of antipeptide antibody (b–d) and pretreated as follows: (b) no pretreatment; (c)  antibody preincubated with a GST–p53/58 tail fusion protein  bound to glutathione Sepharose 4B beads; (d) antibody preincubated with GST-glutathione Sepharose 4B beads as described in  Materials and Methods. In c and d, the unbound fraction was  added to the transport assay.
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Related In: Results  -  Collection

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

Figure 3: Antibody to the p53/58 carboxyl tail inhibits transport from the ER to the Golgi complex. (A) Semiintact NRK cells were prepared and incubated in vitro as described in Materials and Methods. Semiintact cells were incubated with the indicated concentration of antibody (closed squares) or Fab fragments (open squares) for 45 min on ice and then transferred to 32°C for 90 min. (Inset) Preabsorption of the antibody to the cytoplasmic tail neutralizes its inhibitory property. Semiintact cells were incubated in the absence (a) or presence of 5 μg of antipeptide antibody (b–d) and pretreated as follows: (b) no pretreatment; (c) antibody preincubated with a GST–p53/58 tail fusion protein bound to glutathione Sepharose 4B beads; (d) antibody preincubated with GST-glutathione Sepharose 4B beads as described in Materials and Methods. In c and d, the unbound fraction was added to the transport assay.
Mentions: Preincubation of semiintact NRK cells with affinitypurified antibody led to a dose-dependent inhibition of ER to Golgi transport (Fig. 3). The processing of VSV-G to endo H–resistant forms was reduced by 50% in the presence of ∼2 μg antibody with complete inhibition at 8–10 μg (Fig. 3, closed squares). This inhibition was not a consequence of protein aggregation as Fab fragments also inhibited acquisition of endo H resistance at a level comparable to that of the intact antibody (Fig. 3, open squares). To provide further proof that the block in transport was due to the specific neutralization of a p53/58 carboxyl-terminal epitope, antibody was incubated with a GST–p53/58 carboxyl tail fusion protein (GST–p53/58 tail) bound to glutathione Sepharose 4B. The resulting nonabsorbed fraction was tested for activity in the semiintact cell assay. Preincubation of the antibody with the GST–p53/58 tail beads efficiently neutralized its inhibitory effect on protein traffic (Fig. 3, inset, c). In contrast, inhibition was not affected by incubation of antibody with control GST beads that lacked the p53/58 tail (Fig. 3, inset, d). These results show that the antibody blocks transport in NRK cells through a specific interaction with p58 and is the first demonstration that p58 may be required for ER to Golgi transport.

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