Limits...
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.

Show MeSH

Related in: MedlinePlus

Antitail antibody blocks binding of β-COP to a GST fusion protein containing the carboxyl terminus of p53/58 and prevents the recruitment of COPI coats to microsomes. (A) GST  (lanes a–c) or a GST fusion protein containing the carboxyl terminus of p53/58 (lanes d–i) was bound to glutathione Sepharose  4B beads and preincubated in the absence (lanes a–f) or presence  (lanes g–i) of antipeptide antibody as described in Materials and  Methods. Rat liver cytosol was then added and incubated for an  additional 4 h at 4°C. In each case, the unbound fraction (lanes a,  d, and g), the low (75 mM) salt wash (lanes b, e, and h), and the  high (500 mM) salt wash (lanes c, f, and i) were prepared as described in Materials and Methods. The amount of β-COP in each  fraction was determined by Western blotting and densitometry.  (B) Microsomes were prepared from whole cell homogenates as  described in Materials and Methods, mixed with cytosol and 20  μM GTPγS, and either not incubated (lane a) or incubated for 15  min at 37°C (lanes b–g) in the absence (lanes a–c) or presence  (lanes d–g) of the indicated amount of antitail antibody. Membranes were transferred to ice and pelleted, and the amount of  β-COP (arbitrary units) was determined by Western blotting and  densitometry. In lane c, membranes were omitted from the cocktail.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139878&req=5

Figure 9: Antitail antibody blocks binding of β-COP to a GST fusion protein containing the carboxyl terminus of p53/58 and prevents the recruitment of COPI coats to microsomes. (A) GST (lanes a–c) or a GST fusion protein containing the carboxyl terminus of p53/58 (lanes d–i) was bound to glutathione Sepharose 4B beads and preincubated in the absence (lanes a–f) or presence (lanes g–i) of antipeptide antibody as described in Materials and Methods. Rat liver cytosol was then added and incubated for an additional 4 h at 4°C. In each case, the unbound fraction (lanes a, d, and g), the low (75 mM) salt wash (lanes b, e, and h), and the high (500 mM) salt wash (lanes c, f, and i) were prepared as described in Materials and Methods. The amount of β-COP in each fraction was determined by Western blotting and densitometry. (B) Microsomes were prepared from whole cell homogenates as described in Materials and Methods, mixed with cytosol and 20 μM GTPγS, and either not incubated (lane a) or incubated for 15 min at 37°C (lanes b–g) in the absence (lanes a–c) or presence (lanes d–g) of the indicated amount of antitail antibody. Membranes were transferred to ice and pelleted, and the amount of β-COP (arbitrary units) was determined by Western blotting and densitometry. In lane c, membranes were omitted from the cocktail.

Mentions: To assess the possible effect of antibody on coatomer recruitment by p53/58, we analyzed COPI binding to the GST fusion protein containing the carboxyl terminus QQEEAAKKFF residues bound to glutathione Sepharose 4B beads (GST–tail beads). GST–tail beads were incubated with rat liver cytosol, which serves as a rich source of coatomer. After incubation, the beads were washed extensively with either a low (75 mM) or high (500 mM) saltcontaining buffer, and the unbound (low salt wash) and bound-released protein (high salt wash) were analyzed by SDS-PAGE and Western blotting for β-COP. Control beads containing the GST construct alone (minus tail) did not retain β-COP after the high-salt wash (Fig. 9 A, lanes a–c). In contrast, GST–tail beads retained β-COP (Fig. 9 A, lane f). No binding was detected to GST–tail beads in which the di-lysine motif in the carboxyl-tail was mutated to serine residues (not shown). Importantly, preabsorption of the GST–tail beads with the antitail antibody completely blocked β-COP binding (Fig. 9 A, lane i), consistent with the observation that the antibody blocks the recruitment of COPI to membranes in vivo (Fig. 8).


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)

Antitail antibody blocks binding of β-COP to a GST fusion protein containing the carboxyl terminus of p53/58 and prevents the recruitment of COPI coats to microsomes. (A) GST  (lanes a–c) or a GST fusion protein containing the carboxyl terminus of p53/58 (lanes d–i) was bound to glutathione Sepharose  4B beads and preincubated in the absence (lanes a–f) or presence  (lanes g–i) of antipeptide antibody as described in Materials and  Methods. Rat liver cytosol was then added and incubated for an  additional 4 h at 4°C. In each case, the unbound fraction (lanes a,  d, and g), the low (75 mM) salt wash (lanes b, e, and h), and the  high (500 mM) salt wash (lanes c, f, and i) were prepared as described in Materials and Methods. The amount of β-COP in each  fraction was determined by Western blotting and densitometry.  (B) Microsomes were prepared from whole cell homogenates as  described in Materials and Methods, mixed with cytosol and 20  μM GTPγS, and either not incubated (lane a) or incubated for 15  min at 37°C (lanes b–g) in the absence (lanes a–c) or presence  (lanes d–g) of the indicated amount of antitail antibody. Membranes were transferred to ice and pelleted, and the amount of  β-COP (arbitrary units) was determined by Western blotting and  densitometry. In lane c, membranes were omitted from the cocktail.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Antitail antibody blocks binding of β-COP to a GST fusion protein containing the carboxyl terminus of p53/58 and prevents the recruitment of COPI coats to microsomes. (A) GST (lanes a–c) or a GST fusion protein containing the carboxyl terminus of p53/58 (lanes d–i) was bound to glutathione Sepharose 4B beads and preincubated in the absence (lanes a–f) or presence (lanes g–i) of antipeptide antibody as described in Materials and Methods. Rat liver cytosol was then added and incubated for an additional 4 h at 4°C. In each case, the unbound fraction (lanes a, d, and g), the low (75 mM) salt wash (lanes b, e, and h), and the high (500 mM) salt wash (lanes c, f, and i) were prepared as described in Materials and Methods. The amount of β-COP in each fraction was determined by Western blotting and densitometry. (B) Microsomes were prepared from whole cell homogenates as described in Materials and Methods, mixed with cytosol and 20 μM GTPγS, and either not incubated (lane a) or incubated for 15 min at 37°C (lanes b–g) in the absence (lanes a–c) or presence (lanes d–g) of the indicated amount of antitail antibody. Membranes were transferred to ice and pelleted, and the amount of β-COP (arbitrary units) was determined by Western blotting and densitometry. In lane c, membranes were omitted from the cocktail.
Mentions: To assess the possible effect of antibody on coatomer recruitment by p53/58, we analyzed COPI binding to the GST fusion protein containing the carboxyl terminus QQEEAAKKFF residues bound to glutathione Sepharose 4B beads (GST–tail beads). GST–tail beads were incubated with rat liver cytosol, which serves as a rich source of coatomer. After incubation, the beads were washed extensively with either a low (75 mM) or high (500 mM) saltcontaining buffer, and the unbound (low salt wash) and bound-released protein (high salt wash) were analyzed by SDS-PAGE and Western blotting for β-COP. Control beads containing the GST construct alone (minus tail) did not retain β-COP after the high-salt wash (Fig. 9 A, lanes a–c). In contrast, GST–tail beads retained β-COP (Fig. 9 A, lane f). No binding was detected to GST–tail beads in which the di-lysine motif in the carboxyl-tail was mutated to serine residues (not shown). Importantly, preabsorption of the GST–tail beads with the antitail antibody completely blocked β-COP binding (Fig. 9 A, lane i), consistent with the observation that the antibody blocks the recruitment of COPI to membranes in vivo (Fig. 8).

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