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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 specifically inhibit in vitro ER-Golgi  transport. (A) In vitro ER-Golgi transport was performed either  on ice (lane 1) or at 32°C (lanes 2–7) supplemented with the indicated amounts of rbet1 antibodies. (B) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–4)  supplemented with 1.2 μg of rbet1 antibodies (lane 3) or the heat-inactivated antibodies (lane 4). (C) In vitro ER-Golgi transport  was performed either on ice (lane 1) or at 32°C (lanes 2–6) in the  absence (lane 3) or in the presence of rat liver cytosol (rlc) (lanes  1 and 2, and 4–6) supplemented with 0.8 μg of rbet1 antibodies  and indicated amounts of GST-rbet1.
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Figure 11: rbet1 antibodies specifically inhibit in vitro ER-Golgi transport. (A) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–7) supplemented with the indicated amounts of rbet1 antibodies. (B) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–4) supplemented with 1.2 μg of rbet1 antibodies (lane 3) or the heat-inactivated antibodies (lane 4). (C) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–6) in the absence (lane 3) or in the presence of rat liver cytosol (rlc) (lanes 1 and 2, and 4–6) supplemented with 0.8 μg of rbet1 antibodies and indicated amounts of GST-rbet1.

Mentions: The predominant association of rbet1 with the IC and the observation that rbet1 colocalizes with a cargo protein en route to the Golgi suggest that rbet1 may be involved in protein transport from the ER to the Golgi apparatus in mammalian cells. To establish this point, we have examined whether protein transport from the ER to the Golgi could be inhibited by antibodies against rbet1. We adopted the well-established in vitro ER-Golgi transport system using VSV ts045–infected NRK cells (Beckers et al., 1987; Balch et al., 1994). Infected NRK cells were pulse-labeled with [35S]methionine at 40°C so that the labeled G-protein is restricted to the ER. The plasma membrane was then selectively perforated, and the cells were depleted of endogenous cytosol. G-protein can be transported from the ER to the Golgi when these semiintact cells are incubated at permissive temperature (32°C) supplemented with exogenous cytosol and an ATP-regenerating system. ER-Golgi transport was measured by following the extent of conversion of ER-restricted endo H–sensitive G-protein into endo H–resistant Golgi form. As shown in Fig. 11 A, no transport was detected when semiintact cells were incubated on ice (Fig. 11 A, lane 1). The majority of G-protein is converted into endo H–resistant Golgi form when incubated at 32°C (lane 2). Transport from the ER to the Golgi was however inhibited by antibodies against rbet1 in a dose-dependent manner (lanes 3–7). Inhibition was not so obvious when only 0.3 μg of antibodies was added (lane 3). However, ∼60% of the G-protein was not converted into the Golgi form when 0.6 μg of antibodies was added (lane 4). Transport was almost completely inhibited when 0.9 μg (lane 5) or more (lanes 6–7) of antibodies were added. The inhibition is specific because the same amount of heat-denatured antibodies had no effect on transport (compare Fig. 11 A, lane 4, with Fig 11 B, lane 3) and comparable amounts of antibodies against the KDEL receptor (Tang et al. 1997), and several other control antibodies had no effect on ER-Golgi transport of G-protein (data not shown, also see Subramaniam et al., 1996). Furthermore, inhibition exhibited by antibodies against rbet1 could be neutralized by GST-rbet1 (Fig 11 C). G-protein transport to the Golgi is almost completely inhibited by 0.8 μg of rbet1 antibodies (lane 4). However, preincubation of rbet1 antibodies with 0.6 (lane 5) and 1.2 μg (lane 6) of GST-rbet1 resulted in ∼60 and 80% of G-protein being converted into the Golgi form. These results, taken together, suggest that inhibition of ER-Golgi transport by rbet1 antibodies occurs by specific association with endogenous rbet1. rbet1 is therefore essential for ER-Golgi transport.


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 specifically inhibit in vitro ER-Golgi  transport. (A) In vitro ER-Golgi transport was performed either  on ice (lane 1) or at 32°C (lanes 2–7) supplemented with the indicated amounts of rbet1 antibodies. (B) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–4)  supplemented with 1.2 μg of rbet1 antibodies (lane 3) or the heat-inactivated antibodies (lane 4). (C) In vitro ER-Golgi transport  was performed either on ice (lane 1) or at 32°C (lanes 2–6) in the  absence (lane 3) or in the presence of rat liver cytosol (rlc) (lanes  1 and 2, and 4–6) supplemented with 0.8 μg of rbet1 antibodies  and indicated amounts of GST-rbet1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: rbet1 antibodies specifically inhibit in vitro ER-Golgi transport. (A) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–7) supplemented with the indicated amounts of rbet1 antibodies. (B) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–4) supplemented with 1.2 μg of rbet1 antibodies (lane 3) or the heat-inactivated antibodies (lane 4). (C) In vitro ER-Golgi transport was performed either on ice (lane 1) or at 32°C (lanes 2–6) in the absence (lane 3) or in the presence of rat liver cytosol (rlc) (lanes 1 and 2, and 4–6) supplemented with 0.8 μg of rbet1 antibodies and indicated amounts of GST-rbet1.
Mentions: The predominant association of rbet1 with the IC and the observation that rbet1 colocalizes with a cargo protein en route to the Golgi suggest that rbet1 may be involved in protein transport from the ER to the Golgi apparatus in mammalian cells. To establish this point, we have examined whether protein transport from the ER to the Golgi could be inhibited by antibodies against rbet1. We adopted the well-established in vitro ER-Golgi transport system using VSV ts045–infected NRK cells (Beckers et al., 1987; Balch et al., 1994). Infected NRK cells were pulse-labeled with [35S]methionine at 40°C so that the labeled G-protein is restricted to the ER. The plasma membrane was then selectively perforated, and the cells were depleted of endogenous cytosol. G-protein can be transported from the ER to the Golgi when these semiintact cells are incubated at permissive temperature (32°C) supplemented with exogenous cytosol and an ATP-regenerating system. ER-Golgi transport was measured by following the extent of conversion of ER-restricted endo H–sensitive G-protein into endo H–resistant Golgi form. As shown in Fig. 11 A, no transport was detected when semiintact cells were incubated on ice (Fig. 11 A, lane 1). The majority of G-protein is converted into endo H–resistant Golgi form when incubated at 32°C (lane 2). Transport from the ER to the Golgi was however inhibited by antibodies against rbet1 in a dose-dependent manner (lanes 3–7). Inhibition was not so obvious when only 0.3 μg of antibodies was added (lane 3). However, ∼60% of the G-protein was not converted into the Golgi form when 0.6 μg of antibodies was added (lane 4). Transport was almost completely inhibited when 0.9 μg (lane 5) or more (lanes 6–7) of antibodies were added. The inhibition is specific because the same amount of heat-denatured antibodies had no effect on transport (compare Fig. 11 A, lane 4, with Fig 11 B, lane 3) and comparable amounts of antibodies against the KDEL receptor (Tang et al. 1997), and several other control antibodies had no effect on ER-Golgi transport of G-protein (data not shown, also see Subramaniam et al., 1996). Furthermore, inhibition exhibited by antibodies against rbet1 could be neutralized by GST-rbet1 (Fig 11 C). G-protein transport to the Golgi is almost completely inhibited by 0.8 μg of rbet1 antibodies (lane 4). However, preincubation of rbet1 antibodies with 0.6 (lane 5) and 1.2 μg (lane 6) of GST-rbet1 resulted in ∼60 and 80% of G-protein being converted into the Golgi form. These results, taken together, suggest that inhibition of ER-Golgi transport by rbet1 antibodies occurs by specific association with endogenous rbet1. rbet1 is therefore essential for ER-Golgi transport.

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