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 to, and promotes GDP-GTP exchange on, a subset of the Rab GTPases (Burton, 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) of the GTPase that is important for this interaction, protein chimeras were constructed between Rab3a, which binds Mss4, and Rab5a, which does not bind Mss4. We have identified the amino-terminal portion of Rab3a as the Mss4-binding region, with the effector domain being critically 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 Mss4. The region of highest homology between these Rabs, which defines them as members of the same evolutionary branch within the Rab subfamily, coincides with the domain shown here to be critical for Mss4 binding. A mutation in the zinc-binding domain of Mss4 (Mss4 D96H), a region that is highly conserved between Mss4 and its yeast homologue Dss4, completely abolished its property to bind to, and promote GDP-GTP exchange on, Rab3a. Thus, the preservation of the Mss4/Dss4-GTPase interaction appears to have been a critical factor in the evolution of this subset of Rab proteins.
Burton
J L
JL
Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Slepnev
V
V
De Camilli
P V
PV
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017354:Point Mutation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020692:rab3 GTP-Binding Proteins
D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins