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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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Antitail antibody prevents the recruitment of β-COP to  membranes. Uninfected NRK cells grown on coverslips were permeabilized as described (Plutner et al., 1992), incubated at reduced temperature for 80 min at 15°C, and then transferred to  37°C for 20 min in the absence (A) or presence (B) of antitail antibody, and the distribution of β-COP was determined as described in Materials and Methods.
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Figure 8: Antitail antibody prevents the recruitment of β-COP to membranes. Uninfected NRK cells grown on coverslips were permeabilized as described (Plutner et al., 1992), incubated at reduced temperature for 80 min at 15°C, and then transferred to 37°C for 20 min in the absence (A) or presence (B) of antitail antibody, and the distribution of β-COP was determined as described in Materials and Methods.

Mentions: Segregation of p58 from VSV-G during transit through VTCs requires COPI (Aridor et al., 1995). Because COPI components bind to carboxyl-terminal di-lysine and diphenylalanine motifs (Cosson and Letourner, 1994; Fiedler and Simons, 1995; Söhn et al., 1996), the presence of these residues at the cytoplasmic tail of p53/58 suggested that the antibody may inhibit transport by interfering with the binding of coatomer. To address this question morphologically, uninfected, permeabilized NRK cells were preincubated at 15°C for 80 min in the absence of antibody to accumulate VTCs containing p58 (as shown in Fig. 7 A). The cells were then incubated at 37°C for 20 min in the absence (Fig. 8 A) or presence (Fig. 8 B) of antibody. Control cells (lacking antibody) showed a typical intense staining for β-COP in the Golgi region (Duden et al., 1991; Oprins et al., 1993; Aridor et al., 1995). In contrast, cells incubated in the presence of antibody displayed a striking reduction in the number of β-COP–positive structures (Fig. 8 C). A similar reduction in coatomer recruitment in the presence of antibody was observed by incubating cells at 32°C for 30 min without the 15°C preincubation (not shown). The loss of β-COP–positive elements was not observed using a number of other antibodies, including several monoclonal reagents specific for the small GTPases Rab1 (Plutner et al., 1991; Saraste et al., 1995) and Rab2 (Chavrier et al., 1990), which are localized to pre-Golgi intermediates. Moreover, in the case of infected cells, a polyclonal specific to the cytoplasmic tail of VSV-G protein had no effect on the distribution of β-COP–positive elements (not shown). Thus, the anti-p53/58 tail antibody appears to significantly modify, either directly or indirectly, the ability of VTCs located in peripheral and Golgi adjacent sites to recruit COPI.


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 prevents the recruitment of β-COP to  membranes. Uninfected NRK cells grown on coverslips were permeabilized as described (Plutner et al., 1992), incubated at reduced temperature for 80 min at 15°C, and then transferred to  37°C for 20 min in the absence (A) or presence (B) of antitail antibody, and the distribution of β-COP was determined as described in Materials and Methods.
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Related In: Results  -  Collection

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Figure 8: Antitail antibody prevents the recruitment of β-COP to membranes. Uninfected NRK cells grown on coverslips were permeabilized as described (Plutner et al., 1992), incubated at reduced temperature for 80 min at 15°C, and then transferred to 37°C for 20 min in the absence (A) or presence (B) of antitail antibody, and the distribution of β-COP was determined as described in Materials and Methods.
Mentions: Segregation of p58 from VSV-G during transit through VTCs requires COPI (Aridor et al., 1995). Because COPI components bind to carboxyl-terminal di-lysine and diphenylalanine motifs (Cosson and Letourner, 1994; Fiedler and Simons, 1995; Söhn et al., 1996), the presence of these residues at the cytoplasmic tail of p53/58 suggested that the antibody may inhibit transport by interfering with the binding of coatomer. To address this question morphologically, uninfected, permeabilized NRK cells were preincubated at 15°C for 80 min in the absence of antibody to accumulate VTCs containing p58 (as shown in Fig. 7 A). The cells were then incubated at 37°C for 20 min in the absence (Fig. 8 A) or presence (Fig. 8 B) of antibody. Control cells (lacking antibody) showed a typical intense staining for β-COP in the Golgi region (Duden et al., 1991; Oprins et al., 1993; Aridor et al., 1995). In contrast, cells incubated in the presence of antibody displayed a striking reduction in the number of β-COP–positive structures (Fig. 8 C). A similar reduction in coatomer recruitment in the presence of antibody was observed by incubating cells at 32°C for 30 min without the 15°C preincubation (not shown). The loss of β-COP–positive elements was not observed using a number of other antibodies, including several monoclonal reagents specific for the small GTPases Rab1 (Plutner et al., 1991; Saraste et al., 1995) and Rab2 (Chavrier et al., 1990), which are localized to pre-Golgi intermediates. Moreover, in the case of infected cells, a polyclonal specific to the cytoplasmic tail of VSV-G protein had no effect on the distribution of β-COP–positive elements (not shown). Thus, the anti-p53/58 tail antibody appears to significantly modify, either directly or indirectly, the ability of VTCs located in peripheral and Golgi adjacent sites to recruit COPI.

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