SRP-35 A Putative NAD(P)H Binding Protein Of Skeletal Muscle Sarcoplasmic Reticulum Membrane

Abstract

SRP-35 (sarcoplasmic reticulum protein of 35 kDa) is a newly identified integral membrane protein constituent of skeletal muscle sarcoplasmic reticulum. We have deduced the primary structure of the protein from cDNA clones isolated from mouse and rat skeletal muscle cDNA libraries. Primary sequence prediction analysis indicates that the NH2- terminal sequence of SRP-35 encompasses a transmembrane spanning segment or a signal sequence. In addition, SRP-35 is homologous to proteins belonging to the short-chain dehydrogenase/reductases family. Members of this protein family has two domains: the first involved in binding the nucleotide co-factor NAD(P)H, the second responsible for the catalysis of the substrate. SRP35 contains only a putative NAD(P)H binding site. Analysis of tissue distribution of SRP-35 by western blot analysis with affinity purified Ab shows that SRP-35 expression is specific for skeletal muscle since our Ab did not stain any protein in other tissues including heart, brain, liver, kidney, lung, spleen and stomach. Immunohistochemistry of primary cultured mouse myotubes transfected with SRP-35 EGFP construct indicates that SRP-35 is distributed on sarco(endo)plasmic reticulum membranes. Staining of western blot of sarcoplasmic reticulum membrane subfractions isolated from adult mouse skeletal muscle revealed that SRP-35 is associated with heavy sarcoplasmic reticulum. In addition, we found that SRP-35 is an integral membrane protein since it was extracted neither by NaCO3 nor by high salt treatment of isolated sarcoplasmic reticulum membrane, but was solubilised by non-ionic detergent such as CHAPS, DDM and DHPC. We propose that , SRP-35 protein might provide the co-factor to enzymes involved in the generation of local reactive oxygen species within cellular subdomains