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First published online 22 February 2005
doi: 10.1242/jcs.01723
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Research Article |
1 Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Republic of Singapore
2 Office of Life Sciences, MD 11 Level 2, #02-04, 10 Medical Drive, Clinical Research Center, Singapore 117597, Republic of Singapore
3 Membrane Transport Laboratory, Cancer and Cell Biology Division, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, QLD 4029, Australia
* Author for correspondence (e-mail: mcbhwj{at}imcb.a-star.edu.sg)
Accepted 18 January 2005
The TRAPP complex identified in yeast regulates vesicular transport in the early secretory pathway. Although some components of the TRAPP complex are structurally conserved in mammalian cells, the function of the mammalian components has not been examined. We describe our biochemical and functional analysis of mammalian Bet3, the most conserved component of the TRAPP complex. Bet3 mRNA is ubiquitously expressed in all tissues. Antibodies raised against recombinant Bet3 specifically recognize a protein of 22 kDa. In contrast to yeast Bet3p, the majority of Bet3 is present in the cytosol. To investigate the possible involvement of Bet3 in transport events in mammalian cells, we utilized a semi-intact cell system that reconstitutes the transport of the envelope glycoprotein of vesicular stomatitis virus (VSV-G) from the ER to the Golgi apparatus. In this system, antibodies against Bet3 inhibit transport in a dose-dependent manner, and cytosol that is immunodepleted of Bet3 is also defective in this transport. This defect can be rescued by supplementing the Bet3-depleted cytosol with recombinant GST-Bet3. We also show that Bet3 acts after COPII but before Rab1, 
-SNAP and the EGTA-sensitive stage during ER-Golgi transport. Gel filtration analysis demonstrates that Bet3 exists in two distinct pools in the cytosol, the high-molecular-weight pool may represent the TRAPP complex, whereas the other probably represents the monomeric Bet3.
Key words: Tethering complex, TRAPP, SNARE, Bet3, ER, Golgi, Vesicle, Fusion
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