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The mammalian protein (rbet1) homologous to yeast Bet1p is primarily associated with the pre-Golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the Golgi apparatus.

Zhang T, Wong SH, Tang BL, Xu Y, Peter F, Subramaniam VN, Hong W - J. Cell Biol. (1997)

Bottom Line: A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1.In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells.Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 119076, Singapore.

ABSTRACT
Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15 degrees C. However, upon warming up from 15 to 37 degrees C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER-derived vesicles with the cis-Golgi membrane.

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rbet1 antibodies must be present before the EGTA-sensitive stage to exhibit the inhibitory effect on in vitro ER-Golgi transport. In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–11) in the absence (lane 3)  or presence of rat liver cytosol (rlc) (lanes 1 and 2, and 4–11). The  standard transport was performed for lanes 1–4. For lanes 5–11,  transport assay was first performed in the presence of 10 mM  EGTA to arrest the transport at the EGTA-sensitive stage followed by a washing step and second incubation at 32°C to continue the transport. Reagents were supplemented as indicated.
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Figure 12: rbet1 antibodies must be present before the EGTA-sensitive stage to exhibit the inhibitory effect on in vitro ER-Golgi transport. In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–11) in the absence (lane 3) or presence of rat liver cytosol (rlc) (lanes 1 and 2, and 4–11). The standard transport was performed for lanes 1–4. For lanes 5–11, transport assay was first performed in the presence of 10 mM EGTA to arrest the transport at the EGTA-sensitive stage followed by a washing step and second incubation at 32°C to continue the transport. Reagents were supplemented as indicated.

Mentions: In vitro ER-Golgi transport could be inhibited by EGTA at a stage between vesicle docking and fusion (Rexach and Schekman, 1991; Balch et al., 1994; Pind et al., 1994; Aridor et al., 1995; Lupashin et al., 1996; Subramaniam et al., 1996). To gain additional understanding about the involvement of rbet1 in ER-Golgi transport, we have found that rbet1 antibodies must be present before the EGTA-sensitive stage to exhibit an inhibitory effect (Fig. 12). In this experiment, in vitro ER-Golgi transport was first performed in the presence of EGTA to arrest transport at the EGTA-sensitive stage. Semiintact cells were then washed, resuspended in complete transport cocktail, and resumed with a second stage of incubation to continue the events between the EGTA-sensitive stage to the actual membrane fusion. As shown, G-protein remained in the endo H–sensitive ER form after the first stage of incubation (Fig. 12, lane 10). A second incubation in fresh cytosol and complete transport cocktail allowed almost complete conversion of the EGTA-arrested ER form into the endo H–resistant Golgi form (lane 11). Inclusion of rbet1 antibodies in standard transport assay inhibited the transport to the background level (lane 4). However, when rbet1 antibodies were included only in the second stage of incubation, almost complete transport was achieved, suggesting that rbet1 antibodies could not inhibit the transport when included only in the second stage of transport assay. As shown previously (Nuoffer et al., 1994; Aridor et al., 1995), GTP-γ-S (lanes 6–7) and a Rab1 monoclonal antibody (Plutner et al., 1991) (lanes 8–9) were also no longer inhibitory to ER-Golgi transport when supplemented only at the second stage of incubation. These results suggest that rbet1 antibodies can no longer gain access to rbet1 or that rbet1 is no longer required after the EGTA-sensitive stage.


The mammalian protein (rbet1) homologous to yeast Bet1p is primarily associated with the pre-Golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the Golgi apparatus.

Zhang T, Wong SH, Tang BL, Xu Y, Peter F, Subramaniam VN, Hong W - J. Cell Biol. (1997)

rbet1 antibodies must be present before the EGTA-sensitive stage to exhibit the inhibitory effect on in vitro ER-Golgi transport. In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–11) in the absence (lane 3)  or presence of rat liver cytosol (rlc) (lanes 1 and 2, and 4–11). The  standard transport was performed for lanes 1–4. For lanes 5–11,  transport assay was first performed in the presence of 10 mM  EGTA to arrest the transport at the EGTA-sensitive stage followed by a washing step and second incubation at 32°C to continue the transport. Reagents were supplemented as indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140212&req=5

Figure 12: rbet1 antibodies must be present before the EGTA-sensitive stage to exhibit the inhibitory effect on in vitro ER-Golgi transport. In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–11) in the absence (lane 3) or presence of rat liver cytosol (rlc) (lanes 1 and 2, and 4–11). The standard transport was performed for lanes 1–4. For lanes 5–11, transport assay was first performed in the presence of 10 mM EGTA to arrest the transport at the EGTA-sensitive stage followed by a washing step and second incubation at 32°C to continue the transport. Reagents were supplemented as indicated.
Mentions: In vitro ER-Golgi transport could be inhibited by EGTA at a stage between vesicle docking and fusion (Rexach and Schekman, 1991; Balch et al., 1994; Pind et al., 1994; Aridor et al., 1995; Lupashin et al., 1996; Subramaniam et al., 1996). To gain additional understanding about the involvement of rbet1 in ER-Golgi transport, we have found that rbet1 antibodies must be present before the EGTA-sensitive stage to exhibit an inhibitory effect (Fig. 12). In this experiment, in vitro ER-Golgi transport was first performed in the presence of EGTA to arrest transport at the EGTA-sensitive stage. Semiintact cells were then washed, resuspended in complete transport cocktail, and resumed with a second stage of incubation to continue the events between the EGTA-sensitive stage to the actual membrane fusion. As shown, G-protein remained in the endo H–sensitive ER form after the first stage of incubation (Fig. 12, lane 10). A second incubation in fresh cytosol and complete transport cocktail allowed almost complete conversion of the EGTA-arrested ER form into the endo H–resistant Golgi form (lane 11). Inclusion of rbet1 antibodies in standard transport assay inhibited the transport to the background level (lane 4). However, when rbet1 antibodies were included only in the second stage of incubation, almost complete transport was achieved, suggesting that rbet1 antibodies could not inhibit the transport when included only in the second stage of transport assay. As shown previously (Nuoffer et al., 1994; Aridor et al., 1995), GTP-γ-S (lanes 6–7) and a Rab1 monoclonal antibody (Plutner et al., 1991) (lanes 8–9) were also no longer inhibitory to ER-Golgi transport when supplemented only at the second stage of incubation. These results suggest that rbet1 antibodies can no longer gain access to rbet1 or that rbet1 is no longer required after the EGTA-sensitive stage.

Bottom Line: A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1.In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells.Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Membrane Biology Laboratory, Institute of Molecular and Cell Biology, Singapore 119076, Singapore.

ABSTRACT
Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15 degrees C. However, upon warming up from 15 to 37 degrees C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER-derived vesicles with the cis-Golgi membrane.

Show MeSH
Related in: MedlinePlus