Functional dissection of nebulette demonstrates actin binding of nebulin-like repeats and Z-line targeting of SH3 and linker domains.
Nebulette, a 107 kDa protein associated with the I-Z-I complex of cardiac myofibrils, may play an important role in the assembly of the Z-line. Determination of the complete primary structure of 1011 residue human fetal nebulette reveals a four-domain layout similar to skeletal muscle nebulin: a short N-terminal domain, followed by 22 nebulin-like repeats that are linked to a C-terminal Src homology 3 (SH3) domain via a short linker domain. To elucidate the mechanisms of assembly for nebulette in the Z-line, the complete coding sequence or fusions of nebulette domains with green fluorescent protein (GFP) were expressed in cardiomyocytes and fibroblasts. The complete protein localized to Z-lines in cardiac cells and to dense bodies in nonmuscle cells. The GFP-repeat domain forms bundles that are associated with actin filaments in both cell types and disrupts the microfilament network. In contrast, the GFP-repeat plus linker shows limited interaction with dense bodies in nonmuscle cells and the Z-lines of cardiomyocytes. Interestingly, the tagged linker or SH3 is diffusely distributed in nonmuscle cells, but localizes to the Z-lines in cardiomyocytes. Supporting the cellular localization work, recombinant nebulette fragments bind to actin, tropomyosin, and alpha-actinin in in vitro binding assays. These results suggest the repeat domain contains actin binding functions and that the linker domain may target this interaction to Z-lines and dense bodies. Our data also indicate that the linker and SH3 domains can distinguish between dense bodies and Z-lines, suggesting that the ligands for their interactions are specific to these muscular substructures.
Moncman
C L
CL
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, USA.
Wang
K
K
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins; D002478:Cells, Cultured; D002642:Chick Embryo; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D049452:Green Fluorescent Proteins; D006801:Humans; D060588:LIM Domain Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics* / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D060588:LIM Domain Proteins; D008164:Luminescent Proteins; D009124:Muscle Proteins; C486890:NEBL protein, human; D011993:Recombinant Fusion Proteins; C035620:nebulin; D049452:Green Fluorescent Proteins