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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 exists in a protein complex that contains GS28  and syntaxin 5. (A) Golgi extract was immunoprecipitated with  rbet1 antibodies or control antibodies. The immunoprecipitates  were analyzed by immunoblot to detect rbet1, GS28, and syntaxin 5. (B) The immunoprecipitate of rbet1 and control antibodies, together with 100 μg of Golgi extract, were analyzed by immunoblot to detect rbet1 and syntaxin 6.
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Figure 4: rbet1 exists in a protein complex that contains GS28 and syntaxin 5. (A) Golgi extract was immunoprecipitated with rbet1 antibodies or control antibodies. The immunoprecipitates were analyzed by immunoblot to detect rbet1, GS28, and syntaxin 5. (B) The immunoprecipitate of rbet1 and control antibodies, together with 100 μg of Golgi extract, were analyzed by immunoblot to detect rbet1 and syntaxin 6.

Mentions: When the immunoprecipitate of rbet1 antibodies was analyzed by immunoblot, GS28 and syntaxin 5 were detected (Fig. 4 A). The antibodies against syntaxin 5 detect specifically two polypeptides of 38 and 32 kD, respectively (Subramaniam et al., 1997). As shown, the 38-kD form was preferentially coimmunoprecipitated with rbet1, while the 32-kD form was barely detectable. GS28 and syntaxin 5 are two cis-Golgi SNAREs involved in ER-Golgi transport (Banfield et al., 1994; Dascher et al., 1994; Nagahama et al., 1996; Subramaniam et al., 1996). The coimmunoprecipitation of GS28 and syntaxin 5 by rbet1 antibodies is specific because neither GS28 nor syntaxin 5 was detected in the immunoprecipitate of control antibodies. Under identical conditions, syntaxin 6, another Golgi SNARE (Bock et al., 1996), was not coimmunoprecipitated by rbet1 antibodies (Fig. 4 B). In addition, we have observed that rbet1 could also be coimmunoprecipitated by GS28 and syntaxin 5 antibodies (data not shown). These results suggest that rbet1 exists in a protein complex that contains GS28 and syntaxin 5.


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 exists in a protein complex that contains GS28  and syntaxin 5. (A) Golgi extract was immunoprecipitated with  rbet1 antibodies or control antibodies. The immunoprecipitates  were analyzed by immunoblot to detect rbet1, GS28, and syntaxin 5. (B) The immunoprecipitate of rbet1 and control antibodies, together with 100 μg of Golgi extract, were analyzed by immunoblot to detect rbet1 and syntaxin 6.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: rbet1 exists in a protein complex that contains GS28 and syntaxin 5. (A) Golgi extract was immunoprecipitated with rbet1 antibodies or control antibodies. The immunoprecipitates were analyzed by immunoblot to detect rbet1, GS28, and syntaxin 5. (B) The immunoprecipitate of rbet1 and control antibodies, together with 100 μg of Golgi extract, were analyzed by immunoblot to detect rbet1 and syntaxin 6.
Mentions: When the immunoprecipitate of rbet1 antibodies was analyzed by immunoblot, GS28 and syntaxin 5 were detected (Fig. 4 A). The antibodies against syntaxin 5 detect specifically two polypeptides of 38 and 32 kD, respectively (Subramaniam et al., 1997). As shown, the 38-kD form was preferentially coimmunoprecipitated with rbet1, while the 32-kD form was barely detectable. GS28 and syntaxin 5 are two cis-Golgi SNAREs involved in ER-Golgi transport (Banfield et al., 1994; Dascher et al., 1994; Nagahama et al., 1996; Subramaniam et al., 1996). The coimmunoprecipitation of GS28 and syntaxin 5 by rbet1 antibodies is specific because neither GS28 nor syntaxin 5 was detected in the immunoprecipitate of control antibodies. Under identical conditions, syntaxin 6, another Golgi SNARE (Bock et al., 1996), was not coimmunoprecipitated by rbet1 antibodies (Fig. 4 B). In addition, we have observed that rbet1 could also be coimmunoprecipitated by GS28 and syntaxin 5 antibodies (data not shown). These results suggest that rbet1 exists in a protein complex that contains GS28 and syntaxin 5.

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