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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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VSV-G protein accumulates in VTCs in the presence of antibody. NRK cells grown on coverslips were infected with ts045  VSV for 3 h at 39.5°C to accumulate ts045 VSV-G in the ER (Plutner et al., 1992; Balch et al., 1994). The cells were shifted to ice, permeabilized as described in Materials and Methods, and then preincubated in a complete transport cocktail for 45 min on ice in the absence  (A–D) or presence of 10 μg of antitail antibody (E and F). Subsequently, the cells were then either retained on ice (A and B) or shifted  to 32°C for 30 min (C–F) and were transferred to ice to terminate transport and the distribution of VSV-G (A, C, and E) and p58 (B, D,  and F) determined by indirect immunofluorescence as described in Materials and Methods. In control cells (C and D), VSV-G was  transported to the perinuclear Golgi region that partially overlaps with the distribution of p58 (arrowheads). Antibody treatment resulted in the accumulation of VSV-G in punctate VTCs containing p58 (E [VSV-G] and F [p58], arrows).
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Figure 6: VSV-G protein accumulates in VTCs in the presence of antibody. NRK cells grown on coverslips were infected with ts045 VSV for 3 h at 39.5°C to accumulate ts045 VSV-G in the ER (Plutner et al., 1992; Balch et al., 1994). The cells were shifted to ice, permeabilized as described in Materials and Methods, and then preincubated in a complete transport cocktail for 45 min on ice in the absence (A–D) or presence of 10 μg of antitail antibody (E and F). Subsequently, the cells were then either retained on ice (A and B) or shifted to 32°C for 30 min (C–F) and were transferred to ice to terminate transport and the distribution of VSV-G (A, C, and E) and p58 (B, D, and F) determined by indirect immunofluorescence as described in Materials and Methods. In control cells (C and D), VSV-G was transported to the perinuclear Golgi region that partially overlaps with the distribution of p58 (arrowheads). Antibody treatment resulted in the accumulation of VSV-G in punctate VTCs containing p58 (E [VSV-G] and F [p58], arrows).

Mentions: Although it was clear that p58 was required for the transit of VSV-G from VTCs to the Golgi stack, it remained possible that the protein was also essential for export from the ER given its lectin-like properties (Arar et al., 1995; Itin et al., 1996). To address this issue, we used a morphological assay in which NRK cells were infected with ts045 VSV-G for 3 h at 39.5°C (to restrict VSV-G to the ER) (Fig. 6 A) (Plutner et al., 1992). After permeabilization, cells were incubated in the absence or presence of antibody for 45 min on ice and then transferred to 32°C for 30 min, and the distribution of VSV-G was determined by indirect immunofluorescence. Control cells incubated at 32°C in the absence of antibody efficiently transported VSV-G to the juxtanuclear Golgi complex (Fig. 6 C). This distribution overlapped with the typical steady-state distribution of p58 in VTCs localized predominantly to the cisGolgi region (Fig. 6, B and D). In contrast, permeabilized cells incubated in the presence of antibody largely accumulated VSV-G in numerous punctate VTCs scattered throughout the perinuclear and peripheral cytoplasm (Fig. 6 E). VSV-G present in peripheral punctate elements completely overlapped with that of p58 (Fig. 6 F). The antibody had no effect on the distribution of the Golgi complex as assessed by staining with Lens culinaris, which recognizes cis/medial-Golgi compartments (not shown) (Liener et al., 1986; Tisdale et al., 1992). Moreover, cells incubated at 15°C for 80 min in the presence of antibody accumulated VSV-G in pre-Golgi VTCs (not shown). These results demonstrate that the antibody did not affect export in a manner similar to that of the Sar1 GDP-restricted mutant, which prevents COPII assembly and blocks exit of VSV-G from the ER (Kuge et al., 1994; Aridor et al., 1995), but interfered specifically with transit from VTCs to compartments of the Golgi stack. These results are consistent with the lack of processing of VSV-G to endo D– (Fig. 3) and endo H– (Fig. 4) resistant forms in the presence of antibody.


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)

VSV-G protein accumulates in VTCs in the presence of antibody. NRK cells grown on coverslips were infected with ts045  VSV for 3 h at 39.5°C to accumulate ts045 VSV-G in the ER (Plutner et al., 1992; Balch et al., 1994). The cells were shifted to ice, permeabilized as described in Materials and Methods, and then preincubated in a complete transport cocktail for 45 min on ice in the absence  (A–D) or presence of 10 μg of antitail antibody (E and F). Subsequently, the cells were then either retained on ice (A and B) or shifted  to 32°C for 30 min (C–F) and were transferred to ice to terminate transport and the distribution of VSV-G (A, C, and E) and p58 (B, D,  and F) determined by indirect immunofluorescence as described in Materials and Methods. In control cells (C and D), VSV-G was  transported to the perinuclear Golgi region that partially overlaps with the distribution of p58 (arrowheads). Antibody treatment resulted in the accumulation of VSV-G in punctate VTCs containing p58 (E [VSV-G] and F [p58], arrows).
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Related In: Results  -  Collection

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Figure 6: VSV-G protein accumulates in VTCs in the presence of antibody. NRK cells grown on coverslips were infected with ts045 VSV for 3 h at 39.5°C to accumulate ts045 VSV-G in the ER (Plutner et al., 1992; Balch et al., 1994). The cells were shifted to ice, permeabilized as described in Materials and Methods, and then preincubated in a complete transport cocktail for 45 min on ice in the absence (A–D) or presence of 10 μg of antitail antibody (E and F). Subsequently, the cells were then either retained on ice (A and B) or shifted to 32°C for 30 min (C–F) and were transferred to ice to terminate transport and the distribution of VSV-G (A, C, and E) and p58 (B, D, and F) determined by indirect immunofluorescence as described in Materials and Methods. In control cells (C and D), VSV-G was transported to the perinuclear Golgi region that partially overlaps with the distribution of p58 (arrowheads). Antibody treatment resulted in the accumulation of VSV-G in punctate VTCs containing p58 (E [VSV-G] and F [p58], arrows).
Mentions: Although it was clear that p58 was required for the transit of VSV-G from VTCs to the Golgi stack, it remained possible that the protein was also essential for export from the ER given its lectin-like properties (Arar et al., 1995; Itin et al., 1996). To address this issue, we used a morphological assay in which NRK cells were infected with ts045 VSV-G for 3 h at 39.5°C (to restrict VSV-G to the ER) (Fig. 6 A) (Plutner et al., 1992). After permeabilization, cells were incubated in the absence or presence of antibody for 45 min on ice and then transferred to 32°C for 30 min, and the distribution of VSV-G was determined by indirect immunofluorescence. Control cells incubated at 32°C in the absence of antibody efficiently transported VSV-G to the juxtanuclear Golgi complex (Fig. 6 C). This distribution overlapped with the typical steady-state distribution of p58 in VTCs localized predominantly to the cisGolgi region (Fig. 6, B and D). In contrast, permeabilized cells incubated in the presence of antibody largely accumulated VSV-G in numerous punctate VTCs scattered throughout the perinuclear and peripheral cytoplasm (Fig. 6 E). VSV-G present in peripheral punctate elements completely overlapped with that of p58 (Fig. 6 F). The antibody had no effect on the distribution of the Golgi complex as assessed by staining with Lens culinaris, which recognizes cis/medial-Golgi compartments (not shown) (Liener et al., 1986; Tisdale et al., 1992). Moreover, cells incubated at 15°C for 80 min in the presence of antibody accumulated VSV-G in pre-Golgi VTCs (not shown). These results demonstrate that the antibody did not affect export in a manner similar to that of the Sar1 GDP-restricted mutant, which prevents COPII assembly and blocks exit of VSV-G from the ER (Kuge et al., 1994; Aridor et al., 1995), but interfered specifically with transit from VTCs to compartments of the Golgi stack. These results are consistent with the lack of processing of VSV-G to endo D– (Fig. 3) and endo H– (Fig. 4) resistant forms in the presence of antibody.

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