Slac2-a/melanophilin contains multiple PEST-like sequences that are highly sensitive to proteolysis.
The synaptotagmin-like protein homologue lacking C2 domains-a (Slac2-a)/melanophilin was recently identified as the "missing link" between the small GTPase Rab27A and the actin-based motor protein myosin Va. Although formation of a tripartite protein complex by three molecules had been shown to be required for proper melanosome distribution in melanocytes (Kuroda, T. S., Ariga, H., and Fukuda, M. (2003) Mol. Cell. Biol. 23, 5245-5255), the regulatory mechanisms of the complex (i.e. assembly and disassembly of the complex) had never been elucidated. In this study, we discovered that Slac2-a and a closely related isoform, Slac2-c/MyRIP, contain multiple PEST-like sequences (potential signals for rapid protein degradation) in the myosin Va- and actin-binding domains at the C terminus. We found that the C-terminal domain of Slac2-a is highly sensitive to low concentrations of proteases, such as trypsin and calpain, in vitro, whereas the N-terminal Rab27A-binding domain is highly resistant to these proteases. We further found that endogenous calpains selectively cleave Slac2-a, but not Rab27A or myosin Va, in melanocytes. A mutant Slac2-a lacking one of the PEST-like sequences located at the interface between the myosin Va- and actin-binding domains (DeltaPEST; amino acids 399-405) is more stable than the wild-type protein, both in vitro and in melanocytes. Expression of the mutant Slac2-a-DeltaPEST with an N-terminal green fluorescence protein tag often induced perinuclear aggregation of melanosomes ( approximately 40% of the transfected cells) compared with the wild-type Slac2-a. Our findings suggest that protein degradation of Slac2-a is an essential process for proper melanosome distribution in melanocytes.
Fukuda
Mitsunori
M
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
Itoh
Takashi
T
D000199:Actins / Q000737:chemistry; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry*; D005091:Exons; D020558:GTP Phosphohydrolases / Q000737:chemistry; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008544:Melanocytes / Q000378:metabolism; D020460:Melanosomes / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry; D024701:Myosin Type V / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000737:chemistry; D000075745:rab27 GTP-Binding Proteins
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D008164:Luminescent Proteins; C492410:MLPH protein, human; C501262:MYRIP protein, human; D008840:Microfilament Proteins; C435724:Mlph protein, mouse; C513082:Myo5a protein, mouse; D020033:Protein Isoforms; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; D049452:Green Fluorescent Proteins; D014357:Trypsin; D013917:Thrombin; D002154:Calpain; C449002:mu-calpain; D020558:GTP Phosphohydrolases; D024701:Myosin Type V; C512962:Rab27a protein, mouse; D018995:Myosin Heavy Chains; D020691:rab GTP-Binding Proteins