AN EVOLUTIONARILY CONSERVED DOMAIN IN A SUBFAMILY OF RABS IS CRUCIAL FOR THE INTERACTION WITH THE GUANYL NUCLEOTIDE EXCHANGE FACTOR MSS4

Mss4 is a guanine nucleotide exchange factor that specifically binds t o, and promotes GDP-GTP exchange on, a subset of the Rab GTPases (Burt on, J. L., Burns, M. E., Gatti, E., Augustine, G. J., and De Camilli, P. (1994) EMBO J. 13, 5547-5558), In order to identify the domain(s) o f the GTPase that is important for this interaction, protein chimeras were constructed between Rab3a, which binds Mss4, and Rab5a, which doe s not bind Mss4. We have identified the amino-terminal portion of Rab3 a as the Mss4-binding region, with the effector domain being criticall y required for binding and the flanking regions further enhancing the interaction. Sequence comparisons have revealed that Mss4-binding Rabs share more homology with each other than with Rabs that do not bind M ss4. The region of highest homology between these Rabs, which defines them as members of the same evolutionary branch within the Rab subfami ly, coincides with the domain shown here to be critical for Mss4 bindi ng. A mutation in the zinc-binding domain of Mss4 (Mss4 D96H), a regio n that is highly conserved between Mss4 and its yeast homologue Dss4, completely abolished its property to bind to, and promote GDP-GTP exch ange on, Rab3a. Thus, the preservation of the Mss4/Dss4-GTPase interac tion appears to have been a critical factor in the evolution of this s ubset of Rab proteins.