Mammalian Bet3 functions as a cytosolic factor participating in transport from the ER to the Golgi apparatus.
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, alpha-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.
Loh
Eva
E
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Republic of Singapore.
Peter
Frank
F
Subramaniam
V Nathan
VN
Hong
Wanjin
W
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D015152:Blotting, Northern; D022181:COP-Coated Vesicles / Q000737:chemistry; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000378:metabolism; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C079620:SEC13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; C503571:TRAPPC3 protein, human; C503570:TRAPPC3 protein, rat; C496933:Trappc3 protein, mouse; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; D004533:Egtazic Acid; D020761:rab1 GTP-Binding Proteins