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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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NRK cells were treated  with 10 μg/ml nocodazole for 1 h,  fixed, and double-labeled for rbet1  (a and d) with KDEL-R (b) or man  II (e). Also shown are the merged  images (c and f). Bar, 10 μm.
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Figure 6: NRK cells were treated with 10 μg/ml nocodazole for 1 h, fixed, and double-labeled for rbet1 (a and d) with KDEL-R (b) or man II (e). Also shown are the merged images (c and f). Bar, 10 μm.

Mentions: When cells were treated with nocodazole, which is known to fragment the Golgi apparatus (Rogalski and Singer, 1984; Turner and Tartakoff, 1989), rbet1 (Fig. 6, a and d) and the mammalian KDEL receptor (Fig. 6 b) remained predominantly associated with cytoplasmic spotty structures, while the Golgi apparatus marked by mannosidase II became fragmented into several patches (Fig. 6 e). Under this condition, it is interesting to note that a significant portion of the spotty structures marked by rbet1 and the KDEL receptor is no longer colocalized, suggesting that rbet1 and KDEL receptor may reside in distinct structures of the IC. Under this condition, partial colocalization of rbet1 and KDEL receptor in larger dots could be detected, and these structures may either represent the IC or the fragmented Golgi apparatus. Furthermore, it is obvious that the majority of rbet1-containing structures is negative for Golgi mannosidase II labeling (Fig. 6 f). Some rbet1-containing spotty structures are seen in the vicinity of those marked by mannosidase II, although they are not well colocalized, suggesting that this fraction of rbet1 may be associated with a subregion of the fragmented Golgi apparatus that is not enriched in the Golgi mannosidase II.


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)

NRK cells were treated  with 10 μg/ml nocodazole for 1 h,  fixed, and double-labeled for rbet1  (a and d) with KDEL-R (b) or man  II (e). Also shown are the merged  images (c and f). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: NRK cells were treated with 10 μg/ml nocodazole for 1 h, fixed, and double-labeled for rbet1 (a and d) with KDEL-R (b) or man II (e). Also shown are the merged images (c and f). Bar, 10 μm.
Mentions: When cells were treated with nocodazole, which is known to fragment the Golgi apparatus (Rogalski and Singer, 1984; Turner and Tartakoff, 1989), rbet1 (Fig. 6, a and d) and the mammalian KDEL receptor (Fig. 6 b) remained predominantly associated with cytoplasmic spotty structures, while the Golgi apparatus marked by mannosidase II became fragmented into several patches (Fig. 6 e). Under this condition, it is interesting to note that a significant portion of the spotty structures marked by rbet1 and the KDEL receptor is no longer colocalized, suggesting that rbet1 and KDEL receptor may reside in distinct structures of the IC. Under this condition, partial colocalization of rbet1 and KDEL receptor in larger dots could be detected, and these structures may either represent the IC or the fragmented Golgi apparatus. Furthermore, it is obvious that the majority of rbet1-containing structures is negative for Golgi mannosidase II labeling (Fig. 6 f). Some rbet1-containing spotty structures are seen in the vicinity of those marked by mannosidase II, although they are not well colocalized, suggesting that this fraction of rbet1 may be associated with a subregion of the fragmented Golgi apparatus that is not enriched in the Golgi mannosidase II.

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