New N-RAP-binding partners alpha-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly.
N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. To discover more about the role of N-RAP in myofibril assembly, we used the yeast two-hybrid system to screen a mouse skeletal muscle cDNA library for proteins capable of binding N-RAP in a eukaryotic cell. From yeast two-hybrid experiments we were able to identify three new N-RAP binding partners: alpha-actinin, filamin-2, and Krp1 (also called sarcosin). In vitro binding assays were used to verify these interactions and to identify the N-RAP domains involved. Three regions of N-RAP were expressed as His-tagged recombinant proteins, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal LIM domain (N-RAP-LIM), and the region of N-RAP in between the super repeat region and the LIM domain (N-RAP-IB). We detected significant alpha-actinin binding to N-RAP-IB and N-RAP-LIM, filamin binding to N-RAP-SR, and Krp1 binding to N-RAP-SR and N-RAP-IB. During myofibril assembly in cultured chick cardiomyocytes, N-RAP and filamin appear to co-localize with alpha-actinin in the earliest myofibril precursors found near the cell periphery, as well as in the nascent myofibrils that form as these structures fuse laterally. In contrast, Krp1 is not localized until late in the assembly process, when it appears at the periphery of myofibrils that appear to be fusing laterally. The results suggest that sequential recruitment of N-RAP binding partners may serve an important role during myofibril assembly.
Lu
Shajia
S
Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Carroll
Stefanie L
SL
Herrera
Amy H
AH
Ozanne
Bradford
B
Horowits
Robert
R
D000185:Actinin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D064448:Filamins; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology; D009210:Myofibrils / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D002352:Carrier Proteins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; C578661:FLNC protein, human; D064448:Filamins; C586302:KLHL41 protein, human; C405542:Klhl41 protein, rat; D008840:Microfilament Proteins; D009124:Muscle Proteins; C491567:Nrap protein, mouse; D000185:Actinin