The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32.
In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.
Jones
S
S
Department of Biological Sciences, Laboratory for Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Newman
C
C
Liu
F
F
Segev
N
N
D001692:Biological Transport; D002352:Carrier Proteins / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
C098389:BET3 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C406327:transport protein particle, TRAPP; D005982:Glutathione Transferase; C087905:YPT31 protein, S cerevisiae; C060755:YPT1 protein, S cerevisiae; C121633:YPT32 protein, S cerevisiae; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins