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ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage.

Alvarez C, Fujita H, Hubbard A, Sztul E - J. Cell Biol. (1999)

Bottom Line: Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C.Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack.This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

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Mann I relocates from the Golgi to arrested pre-Golgi VTCs. NRK cells grown at 37°C (A–C) or incubated for 3 h at 15°C (D–F) were processed for double label immunofluorescence using anti–Mann I (A and D) and anti–Mann II (B and E) antibodies. In cells grown at 37°C, Mann I colocalized with Mann II in the Golgi region (C), but after 15°C incubation, Mann I relocated to peripheral punctate structures and did not colocalize with the Golgi localized Mann II (F). NRK cells infected with VSVtsO45 were incubated for 2 h at 42°C and for an additional 3 h at 15°C (G–L). Cells were processed for double label immunofluorescence using anti–Mann I (G) and anti–VSV-G protein (I) antibodies, or anti–Mann II (J) and anti–VSV-G protein antibodies (K). Mann I is present in dispersed punctate structures, some of which contain VSV-G protein (I, arrowheads). Mann II remains within the Golgi and does not relocate to peripheral structures containing VSV-G protein (L). NRK cells infected with VSVtsO45 were incubated for 3 h at 42°C, permeabilized, and supplemented with transport cocktails containing complete cytosol (M–O) or p115-depleted cytosol (P–R). After transport at 32°C for 90 min, cells were processed for double label immunofluorescence using anti–Mann I (M and P) and anti–VSV-G protein (N and Q) antibodies. In reactions containing complete cytosol, VSV-G protein is delivered to the Golgi where it colocalizes with Mann I (O). In reactions containing p115-depleted cytosol, Mann I relocates to pre-Golgi VTCs containing arrested VSV-G protein (R, arrowheads). Bar, 10 μm.
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Figure 10: Mann I relocates from the Golgi to arrested pre-Golgi VTCs. NRK cells grown at 37°C (A–C) or incubated for 3 h at 15°C (D–F) were processed for double label immunofluorescence using anti–Mann I (A and D) and anti–Mann II (B and E) antibodies. In cells grown at 37°C, Mann I colocalized with Mann II in the Golgi region (C), but after 15°C incubation, Mann I relocated to peripheral punctate structures and did not colocalize with the Golgi localized Mann II (F). NRK cells infected with VSVtsO45 were incubated for 2 h at 42°C and for an additional 3 h at 15°C (G–L). Cells were processed for double label immunofluorescence using anti–Mann I (G) and anti–VSV-G protein (I) antibodies, or anti–Mann II (J) and anti–VSV-G protein antibodies (K). Mann I is present in dispersed punctate structures, some of which contain VSV-G protein (I, arrowheads). Mann II remains within the Golgi and does not relocate to peripheral structures containing VSV-G protein (L). NRK cells infected with VSVtsO45 were incubated for 3 h at 42°C, permeabilized, and supplemented with transport cocktails containing complete cytosol (M–O) or p115-depleted cytosol (P–R). After transport at 32°C for 90 min, cells were processed for double label immunofluorescence using anti–Mann I (M and P) and anti–VSV-G protein (N and Q) antibodies. In reactions containing complete cytosol, VSV-G protein is delivered to the Golgi where it colocalizes with Mann I (O). In reactions containing p115-depleted cytosol, Mann I relocates to pre-Golgi VTCs containing arrested VSV-G protein (R, arrowheads). Bar, 10 μm.

Mentions: The inhibitory effects of anti–p115 antibodies on the maintenance and reassembly of normal Golgi structure, coupled with the previous finding that p115 is present on VTCs and cycles between the Golgi and earlier secretory compartments (Nelson et al. 1998), suggested a potential role for p115 in ER to Golgi transport. To analyze if p115 is present on functional VTCs transporting cargo from the ER to the Golgi, we used a temperature-sensitive strain of the vesicular stomatitis virus (VSVtsO45) as a transport marker (for review see Bergmann 1989). The viral VSV-G protein fails to exit the ER at 42°C, the nonpermissive temperature, but after shifting the cells to the permissive temperature of 32°C, a wave of VSV-G protein enters the secretory pathway, and its movement from the ER to the Golgi can be monitored morphologically (Pepperkok et al. 1993; Balch et al. 1994). NRK cells infected with the virus and cultured at the nonpermissive temperature for 3 h contain VSV-G protein in the ER, whereas p115 is predominantly detected in the Golgi region (Fig. 4, A–C). When infected cells were subsequently shifted from 42 to 15°C and incubated at 15°C for 3 h, VSV-G protein movement to the Golgi was arrested in peripheral VTCs that also contained p115 (Fig. 4, D–F). We have shown previously that p115 colocalizes with the VTC marker, ERGIC-53 (Schweizer et al. 1988) when VTCs are preferentially accumulated during low (15°C) temperature incubation (Saraste and Svensson 1991; Nelson et al. 1998). Bonafide Golgi proteins such as galactosyl-transferase or Mann II do not redistribute to peripheral VTCs after low temperature treatment (Lippincott-Schwartz et al. 1990; Nelson et al. 1998; see Fig. 10 E). When infected cells were shifted from 42 to 32°C for 1 h, VSV-G protein and p115 colocalized in the Golgi (Fig. 4, G–I). These results indicate that p115 is a component of VTCs that move VSV-G protein from the ER to the Golgi, and raise the possibility that p115 could be involved in VTC dynamics.


ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage.

Alvarez C, Fujita H, Hubbard A, Sztul E - J. Cell Biol. (1999)

Mann I relocates from the Golgi to arrested pre-Golgi VTCs. NRK cells grown at 37°C (A–C) or incubated for 3 h at 15°C (D–F) were processed for double label immunofluorescence using anti–Mann I (A and D) and anti–Mann II (B and E) antibodies. In cells grown at 37°C, Mann I colocalized with Mann II in the Golgi region (C), but after 15°C incubation, Mann I relocated to peripheral punctate structures and did not colocalize with the Golgi localized Mann II (F). NRK cells infected with VSVtsO45 were incubated for 2 h at 42°C and for an additional 3 h at 15°C (G–L). Cells were processed for double label immunofluorescence using anti–Mann I (G) and anti–VSV-G protein (I) antibodies, or anti–Mann II (J) and anti–VSV-G protein antibodies (K). Mann I is present in dispersed punctate structures, some of which contain VSV-G protein (I, arrowheads). Mann II remains within the Golgi and does not relocate to peripheral structures containing VSV-G protein (L). NRK cells infected with VSVtsO45 were incubated for 3 h at 42°C, permeabilized, and supplemented with transport cocktails containing complete cytosol (M–O) or p115-depleted cytosol (P–R). After transport at 32°C for 90 min, cells were processed for double label immunofluorescence using anti–Mann I (M and P) and anti–VSV-G protein (N and Q) antibodies. In reactions containing complete cytosol, VSV-G protein is delivered to the Golgi where it colocalizes with Mann I (O). In reactions containing p115-depleted cytosol, Mann I relocates to pre-Golgi VTCs containing arrested VSV-G protein (R, arrowheads). Bar, 10 μm.
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Figure 10: Mann I relocates from the Golgi to arrested pre-Golgi VTCs. NRK cells grown at 37°C (A–C) or incubated for 3 h at 15°C (D–F) were processed for double label immunofluorescence using anti–Mann I (A and D) and anti–Mann II (B and E) antibodies. In cells grown at 37°C, Mann I colocalized with Mann II in the Golgi region (C), but after 15°C incubation, Mann I relocated to peripheral punctate structures and did not colocalize with the Golgi localized Mann II (F). NRK cells infected with VSVtsO45 were incubated for 2 h at 42°C and for an additional 3 h at 15°C (G–L). Cells were processed for double label immunofluorescence using anti–Mann I (G) and anti–VSV-G protein (I) antibodies, or anti–Mann II (J) and anti–VSV-G protein antibodies (K). Mann I is present in dispersed punctate structures, some of which contain VSV-G protein (I, arrowheads). Mann II remains within the Golgi and does not relocate to peripheral structures containing VSV-G protein (L). NRK cells infected with VSVtsO45 were incubated for 3 h at 42°C, permeabilized, and supplemented with transport cocktails containing complete cytosol (M–O) or p115-depleted cytosol (P–R). After transport at 32°C for 90 min, cells were processed for double label immunofluorescence using anti–Mann I (M and P) and anti–VSV-G protein (N and Q) antibodies. In reactions containing complete cytosol, VSV-G protein is delivered to the Golgi where it colocalizes with Mann I (O). In reactions containing p115-depleted cytosol, Mann I relocates to pre-Golgi VTCs containing arrested VSV-G protein (R, arrowheads). Bar, 10 μm.
Mentions: The inhibitory effects of anti–p115 antibodies on the maintenance and reassembly of normal Golgi structure, coupled with the previous finding that p115 is present on VTCs and cycles between the Golgi and earlier secretory compartments (Nelson et al. 1998), suggested a potential role for p115 in ER to Golgi transport. To analyze if p115 is present on functional VTCs transporting cargo from the ER to the Golgi, we used a temperature-sensitive strain of the vesicular stomatitis virus (VSVtsO45) as a transport marker (for review see Bergmann 1989). The viral VSV-G protein fails to exit the ER at 42°C, the nonpermissive temperature, but after shifting the cells to the permissive temperature of 32°C, a wave of VSV-G protein enters the secretory pathway, and its movement from the ER to the Golgi can be monitored morphologically (Pepperkok et al. 1993; Balch et al. 1994). NRK cells infected with the virus and cultured at the nonpermissive temperature for 3 h contain VSV-G protein in the ER, whereas p115 is predominantly detected in the Golgi region (Fig. 4, A–C). When infected cells were subsequently shifted from 42 to 15°C and incubated at 15°C for 3 h, VSV-G protein movement to the Golgi was arrested in peripheral VTCs that also contained p115 (Fig. 4, D–F). We have shown previously that p115 colocalizes with the VTC marker, ERGIC-53 (Schweizer et al. 1988) when VTCs are preferentially accumulated during low (15°C) temperature incubation (Saraste and Svensson 1991; Nelson et al. 1998). Bonafide Golgi proteins such as galactosyl-transferase or Mann II do not redistribute to peripheral VTCs after low temperature treatment (Lippincott-Schwartz et al. 1990; Nelson et al. 1998; see Fig. 10 E). When infected cells were shifted from 42 to 32°C for 1 h, VSV-G protein and p115 colocalized in the Golgi (Fig. 4, G–I). These results indicate that p115 is a component of VTCs that move VSV-G protein from the ER to the Golgi, and raise the possibility that p115 could be involved in VTC dynamics.

Bottom Line: Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C.Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack.This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

Show MeSH
Related in: MedlinePlus