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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 is a 17-kD protein enriched in the membrane  fraction of the Golgi and the IC. (A) Total membrane fraction of  NRK cells was resolved by SDS-PAGE and transferred to a filter. The filter was incubated with rbet1 antibodies in the absence  (lane 2) or presence of GST-rbet1 (lane 3). (B) Total membrane  (TM), microsomal membrane (MM), and Golgi membrane (GM)  fractions derived from rat liver were analyzed by immunoblot to  detect rbet1, α2,6-sialyltransferase (ST), and the IC-enriched  protein p58.
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Figure 2: rbet1 is a 17-kD protein enriched in the membrane fraction of the Golgi and the IC. (A) Total membrane fraction of NRK cells was resolved by SDS-PAGE and transferred to a filter. The filter was incubated with rbet1 antibodies in the absence (lane 2) or presence of GST-rbet1 (lane 3). (B) Total membrane (TM), microsomal membrane (MM), and Golgi membrane (GM) fractions derived from rat liver were analyzed by immunoblot to detect rbet1, α2,6-sialyltransferase (ST), and the IC-enriched protein p58.

Mentions: When the total membrane fraction derived from NRK cells was analyzed by immunoblot using rbet1 antibodies, a major polypeptide of about 17-kD was detected (Fig. 2 A, lane 2). Detection of this polypeptide was abolished by preincubation of antibodies with GST-rbet1 (Fig. 2 A, lane 3) but not with GST (data not shown), demonstrating that the 17-kD polypeptide is rbet1. When rat liver total membranes, microsomal membranes, and Golgi membranes (GM) were analyzed by immunoblot, rbet1 was enriched in the GM fraction (Fig. 2 B, upper panel). Markers of the IC are also known to be enriched in the GM fraction when separated by sucrose gradient (Saraste et al., 1987; Schweizer et al., 1988). As shown, similar to α2,6-sialyltransferase (Fig. 2 B, middle panel), the IC marker p58 is also enriched, although to a lesser extent, in the GM fraction (Fig. 2 B, lower panel).


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 is a 17-kD protein enriched in the membrane  fraction of the Golgi and the IC. (A) Total membrane fraction of  NRK cells was resolved by SDS-PAGE and transferred to a filter. The filter was incubated with rbet1 antibodies in the absence  (lane 2) or presence of GST-rbet1 (lane 3). (B) Total membrane  (TM), microsomal membrane (MM), and Golgi membrane (GM)  fractions derived from rat liver were analyzed by immunoblot to  detect rbet1, α2,6-sialyltransferase (ST), and the IC-enriched  protein p58.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: rbet1 is a 17-kD protein enriched in the membrane fraction of the Golgi and the IC. (A) Total membrane fraction of NRK cells was resolved by SDS-PAGE and transferred to a filter. The filter was incubated with rbet1 antibodies in the absence (lane 2) or presence of GST-rbet1 (lane 3). (B) Total membrane (TM), microsomal membrane (MM), and Golgi membrane (GM) fractions derived from rat liver were analyzed by immunoblot to detect rbet1, α2,6-sialyltransferase (ST), and the IC-enriched protein p58.
Mentions: When the total membrane fraction derived from NRK cells was analyzed by immunoblot using rbet1 antibodies, a major polypeptide of about 17-kD was detected (Fig. 2 A, lane 2). Detection of this polypeptide was abolished by preincubation of antibodies with GST-rbet1 (Fig. 2 A, lane 3) but not with GST (data not shown), demonstrating that the 17-kD polypeptide is rbet1. When rat liver total membranes, microsomal membranes, and Golgi membranes (GM) were analyzed by immunoblot, rbet1 was enriched in the GM fraction (Fig. 2 B, upper panel). Markers of the IC are also known to be enriched in the GM fraction when separated by sucrose gradient (Saraste et al., 1987; Schweizer et al., 1988). As shown, similar to α2,6-sialyltransferase (Fig. 2 B, middle panel), the IC marker p58 is also enriched, although to a lesser extent, in the GM fraction (Fig. 2 B, lower panel).

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