IDENTIFICATION OF YEAST COMPONENT-A - RECONSTITUTION OF THE GERANYLGERANYLTRANSFERASE THAT MODIFIES YPT1P AND SEC4P

Members of a large family of small GTP-binding proteins, termed Rabs i n mammalian cells or Ypt and Sec4 in yeast, regulate vesicular traffic in all eukaryotic cells. These proteins are able to bind to membranes because they are modified by the type II geranylgeranyltransferase (G GTase-II), a multisubunit complex. Component A, encoded by the choroid eremia gene in humans, is an escort protein that brings Rabs to compon ent B, the catalytic alpha/beta heterodimer. Mutations in the catalyti c subunits of the yeast GGTase-II (Bet2p/Mad2p) disrupt the membrane a ttachment of Ypt1p and Sec4p and this in turn blocks membrane traffic. In mammalian cells, deletions in choroideremia lead only to retinal d egeneration, even though GGTase-II activity is defective. The yeast MR S6 gene encodes a protein that is approximate to 30% identical to the choroideremia gene product. Here we show that the addition of recombin ant Mrs6p to bacterially expressed Bet2p (beta subunit) and Mad2p (alp ha subunit) reconstitutes GGTase-II activity in vitro, demonstrating t hat Mrs6p is yeast component A. Like Bet2p and Mad2p, Mrs6p is require d for the membrane attachment of Ypt1p and Sec4p in vivo. In contrast to what has been observed before for the loss of function of the choro ideremia gene, the depletion of Mrs6p from yeast cells blocks vesicula r transport. Thus, these findings suggest that there is one essential escort protein in yeast, while more than one may exist in mammalian ce lls.