Molecular cloning, characterization and expression of a novel family of tail-anchored membrane proteins from the myocardium.

Abstract

The process of Excitation-Contraction (E-C) coupling in cardiac muscle is dependent on calcium. Many proteins that regulate calcium homoeostasis in the cardiocyte are also calcium binding proteins. Two cDNAs were previously isolated by screening a rabbit heart cDNA expression library with an antiserum that recognized two calcium binding polypeptides of the sarcolemma. These cDNAs and other related cDNAs, which were isolated from cardiac cDNA libraries, were shown to encode a novel family of sarcolemmal associated proteins (SLAPs). The SLAP cDNAs had divergent $5\sp\prime$ termini and could potentially encode polypeptides of 37, 46, and 74 kDa molecular designated SLAP 1, SLAP2 and SLAP3 respectively. Three SLAP transcripts of 5.9, 4.6 and 3.5 kb were found in heart. The 5.9 kb transcript was ubiquitously expressed while the 3.5 kb and 4.6 kb transcripts were predominantly expressed in cardiac, soleus and smooth muscle. SLAP was encoded by a single gene that mapped to chromosome 3p14.3-21.2 and thus the various transcripts were likely generated by alternative splicing or alternative promoter usage. The primary structure of SLAP predicted that it would have large regions of coiled-coil structure, which included an eleven heptad acidic amphipathic a-helical segment containing two leucine zippers, and a carboxyl terminal transmembrane domain. Three SLAPs of 81, 45 and 35 kDa were shown to be associated with the cardiac sarcolemmal membranes. SLAPs could only be solubilized from cardiac membrane with detergents suggesting that they were integral membrane proteins. Immunohistochemical localization of SLAP in cardiac muscle revealed that SLAP associated with the sarcolemma and displayed a reticular pattern of staining that resembled the transverse-tubules and/or the sarcoplasmic reticulum. Expression studies showed that MYC-epitope tagged SLAP localized to regions of juxtaposition between neighbouring cell membranes although an intracellular pool of the protein was also present in cells undergoing apparent cleavage. SLAPs appeared to be the mammalian orthologues of $\rm TOP\sb{AP},$ a topographically graded protein expressed in the developing chick retina and tectum (Savitt et al., 1995): Immunofluorescence studies with specific antibodies raised against SLAP fusion proteins showed that SLAP expression was developmentally regulated in the rat retina where it is enriched in Mueller glial cells. However, no evidence of a topographical gradient of SLAP distribution was found in either the mammalian retina, superior colliculus or the lateral geniculate nucleus. In the neonatal rat brain SLAP was selectively expressed in neurons of the hippocampus as well as in the support cells of the nerve fiber layer of the olfactory bulb. SLAP also shared 44% homology with USO1, a yeast protein involved in the fusion of vesicles between the endoplasmic reticulum and the Golgi (Sapperstein et al., 1996). SLAP expression was found to inhibit the fusion of myoblast fusion without effecting their differentiation. In view of the role of tail-anchored-membrane proteins in membrane fusion and the similarity of SLAPs to USO1, we propose that SLAPs may define a novel family of proteins involved in mediating membrane-membrane interactions.