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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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The mammalian bet1 proteins are highly conserved.  The amino acid sequences of human, rat, and mouse bet1 are  aligned and residues identical among them are shaded.
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Figure 1: The mammalian bet1 proteins are highly conserved. The amino acid sequences of human, rat, and mouse bet1 are aligned and residues identical among them are shaded.

Mentions: Searching the EST database with the yeast Bet1p sequence led to the identification of an EST clone (accession number R52442) encoding a putative human homologue. During the course of our study, a rat homologue (rbet1) was published (Hay et al., 1996), and more EST clones for the human (accession numbers AA305708, AA112610, AA305267, and W84841) as well as for mouse (accession numbers AA245530, W70983, and W18376) homologues were subsequently identified in the database. The human and mouse EST clones were sequenced to obtain the coding nucleotide sequence and hence the amino acid sequences of human and mouse bet1. As aligned in Fig. 1, human, rat, and mouse bet1 (hbet1, rbet1, and mbet1, respectively) are highly homologous (hbet1 is ∼93% identical to rbet and mbet1, while rbet1 and mbet1 share over 98% identity). All the mammalian homologues are ∼20% identical to Bet1p and share an overall amino acid sequence similarity of about 38–40% with Bet1p. The recombinant cytoplasmic domain of hbet1 was initially expressed as a fusion protein to GST (GST-hbet1) and was used to immunize rabbits. Polyclonal antibodies against hbet1, however, cross-react poorly with rbet1 in NRK cells, despite the fact that hbet1 and rbet1 are highly homologous. To facilitate our morphological and functional studies in NRK cells, we subsequently expressed the cytoplasmic region (residues 1–81) of rbet1 (GST-rbet1), and affinity-purified rabbit antibodies against GST-rbet1 were used in all subsequent experiments.


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)

The mammalian bet1 proteins are highly conserved.  The amino acid sequences of human, rat, and mouse bet1 are  aligned and residues identical among them are shaded.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The mammalian bet1 proteins are highly conserved. The amino acid sequences of human, rat, and mouse bet1 are aligned and residues identical among them are shaded.
Mentions: Searching the EST database with the yeast Bet1p sequence led to the identification of an EST clone (accession number R52442) encoding a putative human homologue. During the course of our study, a rat homologue (rbet1) was published (Hay et al., 1996), and more EST clones for the human (accession numbers AA305708, AA112610, AA305267, and W84841) as well as for mouse (accession numbers AA245530, W70983, and W18376) homologues were subsequently identified in the database. The human and mouse EST clones were sequenced to obtain the coding nucleotide sequence and hence the amino acid sequences of human and mouse bet1. As aligned in Fig. 1, human, rat, and mouse bet1 (hbet1, rbet1, and mbet1, respectively) are highly homologous (hbet1 is ∼93% identical to rbet and mbet1, while rbet1 and mbet1 share over 98% identity). All the mammalian homologues are ∼20% identical to Bet1p and share an overall amino acid sequence similarity of about 38–40% with Bet1p. The recombinant cytoplasmic domain of hbet1 was initially expressed as a fusion protein to GST (GST-hbet1) and was used to immunize rabbits. Polyclonal antibodies against hbet1, however, cross-react poorly with rbet1 in NRK cells, despite the fact that hbet1 and rbet1 are highly homologous. To facilitate our morphological and functional studies in NRK cells, we subsequently expressed the cytoplasmic region (residues 1–81) of rbet1 (GST-rbet1), and affinity-purified rabbit antibodies against GST-rbet1 were used in all subsequent experiments.

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