Obscurin is a ligand for small ankyrin 1 in skeletal muscle.
The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.
Kontrogianni-Konstantopoulos
Aikaterini
A
Department of Physiology, University of Maryland, School of Medicine, Baltimore 21201, USA. akons001@umaryland.edu
Jones
Ellene M
EM
Van Rossum
Damian B
DB
Bloch
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002458:Cell Fractionation; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
C110202:ANK1 protein, human; D017487:Ankyrins; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; D009124:Muscle Proteins; C503780:Obscn protein, rat; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C578224:OBSCN protein, human; D017346:Protein Serine-Threonine Kinases