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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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Antipeptide antibody identifies a protein with a p53/58like distribution localized to VTCs in peripheral and Golgi adjacent sites. (A) HeLa, (B) NRK, and (C) BHK cells were plated  on coverslips and cultured overnight. The cells were fixed, permeabilized, and stained with affinity-purified polyclonal antibody  to the cytoplasmic domain of p53/58, followed by FITC-goat anti– rabbit as described in Materials and Methods. In all three cell  types, punctate elements near the Golgi complex were labeled by  the antibody. This staining pattern is similar to that observed with  antibodies that recognize the luminal domain of p53 and p58  (Schweizer et al., 1990; Saraste and Svensson, 1991).
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Figure 2: Antipeptide antibody identifies a protein with a p53/58like distribution localized to VTCs in peripheral and Golgi adjacent sites. (A) HeLa, (B) NRK, and (C) BHK cells were plated on coverslips and cultured overnight. The cells were fixed, permeabilized, and stained with affinity-purified polyclonal antibody to the cytoplasmic domain of p53/58, followed by FITC-goat anti– rabbit as described in Materials and Methods. In all three cell types, punctate elements near the Golgi complex were labeled by the antibody. This staining pattern is similar to that observed with antibodies that recognize the luminal domain of p53 and p58 (Schweizer et al., 1990; Saraste and Svensson, 1991).

Mentions: To extend these results, an ELISA was performed to define the epitope recognized by the antibody. As shown in Table I, the antibody recognized a peptide that coded for the wild-type p53/58 cytoplasmic tail (QQEAAAKKFF) or a GST fusion protein containing the cytoplasmic tail of p53/58 with similar affinity. Moreover, the GST–peptide fusion proteins containing FIDEKKMP or KAHKSKTH, which were not recognized by antitail antibody on Western blots (Fig. 2 C), were similarly unreactive when bound to microtitre wells. Removal of the QQE residues did not affect antibody recognition (Table I), indicating that the epitope responsible for binding was in the terminal seven residues. Mutation of the di-lysine residues to serine had only a partial effect on antibody binding (Table I). However, mutation of the terminal FF residues to either AA or MP completely abrogated antibody recognition (Table I). In contrast, a peptide corresponding to the tail of p23/24c (RRFFKAKKLIE) (Fielder et al., 1996; Söhn et al., 1996), which contains an internal FF motif, was not recognized (Table 1). The combined results indicate that the dominant epitope recognized by the affinity-purified antitail antibody requires terminal FF residues with a weaker contribution of adjacent di-lysine residues. Importantly, neither di-lysine nor internal di-phenylalanine residues are sufficient to elicit antibody recognition.


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)

Antipeptide antibody identifies a protein with a p53/58like distribution localized to VTCs in peripheral and Golgi adjacent sites. (A) HeLa, (B) NRK, and (C) BHK cells were plated  on coverslips and cultured overnight. The cells were fixed, permeabilized, and stained with affinity-purified polyclonal antibody  to the cytoplasmic domain of p53/58, followed by FITC-goat anti– rabbit as described in Materials and Methods. In all three cell  types, punctate elements near the Golgi complex were labeled by  the antibody. This staining pattern is similar to that observed with  antibodies that recognize the luminal domain of p53 and p58  (Schweizer et al., 1990; Saraste and Svensson, 1991).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Antipeptide antibody identifies a protein with a p53/58like distribution localized to VTCs in peripheral and Golgi adjacent sites. (A) HeLa, (B) NRK, and (C) BHK cells were plated on coverslips and cultured overnight. The cells were fixed, permeabilized, and stained with affinity-purified polyclonal antibody to the cytoplasmic domain of p53/58, followed by FITC-goat anti– rabbit as described in Materials and Methods. In all three cell types, punctate elements near the Golgi complex were labeled by the antibody. This staining pattern is similar to that observed with antibodies that recognize the luminal domain of p53 and p58 (Schweizer et al., 1990; Saraste and Svensson, 1991).
Mentions: To extend these results, an ELISA was performed to define the epitope recognized by the antibody. As shown in Table I, the antibody recognized a peptide that coded for the wild-type p53/58 cytoplasmic tail (QQEAAAKKFF) or a GST fusion protein containing the cytoplasmic tail of p53/58 with similar affinity. Moreover, the GST–peptide fusion proteins containing FIDEKKMP or KAHKSKTH, which were not recognized by antitail antibody on Western blots (Fig. 2 C), were similarly unreactive when bound to microtitre wells. Removal of the QQE residues did not affect antibody recognition (Table I), indicating that the epitope responsible for binding was in the terminal seven residues. Mutation of the di-lysine residues to serine had only a partial effect on antibody binding (Table I). However, mutation of the terminal FF residues to either AA or MP completely abrogated antibody recognition (Table I). In contrast, a peptide corresponding to the tail of p23/24c (RRFFKAKKLIE) (Fielder et al., 1996; Söhn et al., 1996), which contains an internal FF motif, was not recognized (Table 1). The combined results indicate that the dominant epitope recognized by the affinity-purified antitail antibody requires terminal FF residues with a weaker contribution of adjacent di-lysine residues. Importantly, neither di-lysine nor internal di-phenylalanine residues are sufficient to elicit antibody recognition.

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