Distinct Rab27A binding affinities of Slp2-a and Slac2-a/melanophilin: Hierarchy of Rab27A effectors.
The small GTPase Rab27A has recently been shown to regulate melanosome transport in mammalian skin melanocytes through sequentially interacting with two Rab27A effectors, Slac2-a/melanophilin and Slp2-a. Although Slac2-a and Slp2-a contain a similar N-terminal Rab27A-binding domain (named SHD, Slp homology domain), nothing is known about the functional differences between the Slac2-a SHD and Slp2-a SHD. In this study, the Rab27A-binding affinity of ten putative Rab27A effector proteins has been investigated. It has been found that they could be classified into a low-affinity group (e.g., Slac2-a) and a high-affinity group (e.g., Slp2-a and Slp4-a) based on their Rab27A-binding affinity. Kinetic analysis of the GTP-Rab27A-binding to the SHD of Slp2-a, Slp4-a, and Slac2-a by surface plasmon resonance further indicated that the kinetic parameters of Rab27A for the Slp2-a SHD, Slp4-a SHD, and Slac2-a SHD consisted of a fast association rate constant (3.35 x 10(4), 2.06 x 10(4), and 2.11 x 10(4) M(-1) s(-1), respectively) and a slow dissociation rate constant (4.48 x 10(-4), 3.96 x 10(-4), and 2.37 x 10(-3) s(-1) respectively), and their equilibrium dissociation constants were determined to be 13.4, 19.2, and 112 nM, respectively. Our data suggest that distinct Rab27A binding activities of Slac2-a and Slp2-a ensure the order (or hierarchy) of Rab27A effectors that sequentially function in melanosome transport in melanocytes.
Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
Mitsunori
M
Fukuda
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D007700:Kinetics; D011485:Protein Binding; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C492410:MLPH protein, human; C512779:SYTL4 protein, human; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; C512962:Rab27a protein, mouse; D020691:rab GTP-Binding Proteins