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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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Double-labeling of rbet1 with ERGIC-53 in control  cells (A and B), cells pretreated with 10 μg/ml brefeldin A for 1 h  (C and D), and cells preincubated at 15°C for 3 h (E and F). Bar,  10 μm.
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Figure 8: Double-labeling of rbet1 with ERGIC-53 in control cells (A and B), cells pretreated with 10 μg/ml brefeldin A for 1 h (C and D), and cells preincubated at 15°C for 3 h (E and F). Bar, 10 μm.

Mentions: To provide additional evidence for the preferential association of rbet1 with the IC, we have double-labeled cells with antibodies against rbet1 and a monoclonal antibody against ERGIC-53, which is normally enriched in the IC and cycles preferentially between the IC and the ER (Schweizer et al. 1988, 1990). Because only polyclonal antibodies against rbet1 and monoclonal antibody specific for human ERGIC-53/p58 are available, this experiment was only performed in a human cell line (HeLa). As shown in Fig. 8, at steady state rbet1 and ERGIC-53 are colocalized well in spotty structures enriched in the Golgi region (Fig. 8, A and B). In cells pretreated with brefeldin A, they are similarly colocalized in the spotty structures enriched in the perinuclear region (Fig. 8, C and D). When cells were preincubated at 15°C, rbet1 and ERGIC-53 could be seen to concentrate in a compact Golgi-like structure, although rbet1 could be also seen in peripheral spotty structures while association of ERGIC-53 with the ER became more obvious. ERGIC-53 has also been shown previously to shift to a compact Golgi-like structure when cells are preincubated at 15°C (Lippincott-Schwartz et al., 1990). The effect of 15°C preincubation on the IC markers in HeLa cells is different from that observed in NRK cells, in which the IC markers became more enriched in peripheral spotty structures as shown above. These results further support our interpretation that rbet1 is preferentially associated with the IC.


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)

Double-labeling of rbet1 with ERGIC-53 in control  cells (A and B), cells pretreated with 10 μg/ml brefeldin A for 1 h  (C and D), and cells preincubated at 15°C for 3 h (E and F). Bar,  10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Double-labeling of rbet1 with ERGIC-53 in control cells (A and B), cells pretreated with 10 μg/ml brefeldin A for 1 h (C and D), and cells preincubated at 15°C for 3 h (E and F). Bar, 10 μm.
Mentions: To provide additional evidence for the preferential association of rbet1 with the IC, we have double-labeled cells with antibodies against rbet1 and a monoclonal antibody against ERGIC-53, which is normally enriched in the IC and cycles preferentially between the IC and the ER (Schweizer et al. 1988, 1990). Because only polyclonal antibodies against rbet1 and monoclonal antibody specific for human ERGIC-53/p58 are available, this experiment was only performed in a human cell line (HeLa). As shown in Fig. 8, at steady state rbet1 and ERGIC-53 are colocalized well in spotty structures enriched in the Golgi region (Fig. 8, A and B). In cells pretreated with brefeldin A, they are similarly colocalized in the spotty structures enriched in the perinuclear region (Fig. 8, C and D). When cells were preincubated at 15°C, rbet1 and ERGIC-53 could be seen to concentrate in a compact Golgi-like structure, although rbet1 could be also seen in peripheral spotty structures while association of ERGIC-53 with the ER became more obvious. ERGIC-53 has also been shown previously to shift to a compact Golgi-like structure when cells are preincubated at 15°C (Lippincott-Schwartz et al., 1990). The effect of 15°C preincubation on the IC markers in HeLa cells is different from that observed in NRK cells, in which the IC markers became more enriched in peripheral spotty structures as shown above. These results further support our interpretation that rbet1 is preferentially associated with the IC.

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