Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein

Methods Enzymol. 2005;403:431-44. doi: 10.1016/S0076-6879(05)03038-7.

Abstract

Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • COS Cells
  • Carrier Proteins / metabolism*
  • Chlorocebus aethiops
  • Dyneins / metabolism*
  • Mice
  • Microfilament Proteins / metabolism*
  • Myosin Heavy Chains / metabolism*
  • Myosin Type V / metabolism*
  • Myosin VIIa
  • Myosins / metabolism*
  • PC12 Cells
  • Rats
  • rab GTP-Binding Proteins / metabolism*
  • rab27 GTP-Binding Proteins

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Microfilament Proteins
  • Mlph protein, mouse
  • Myo5a protein, mouse
  • Myo5a protein, rat
  • Myo7a protein, mouse
  • Myo7a protein, rat
  • Myosin VIIa
  • rab27 GTP-Binding Proteins
  • Myosin Type V
  • Rab27a protein, mouse
  • Rab27a protein, rat
  • Myosin Heavy Chains
  • Myosins
  • Dyneins
  • rab GTP-Binding Proteins