THE INTRACOMPARTMENTAL SORTING OF MYOSIN ALKALI LIGHT-CHAIN ISOPROTEINS REFLECTS THE SEQUENCE OF DEVELOPMENTAL EXPRESSION AS DETERMINED BY DOUBLE EPITOPE-TAGGING COMPETITION

In order to compare within the same cell the various degrees of specif icity of myosin alkali light chain (MLC) isoproteins sorting to sarcom eres, a competition assay was established using double epitope tagging , Various combinations of two different MLC isoform cDNAs tagged with either a vesicular stomatitis virus VSV-G (VSV) or a medium T (mT) pro tein epitope were co-expressed in cultured cardiomyocytes from adult a nd neonatal rat ventricles, Expressed isoproteins were detected by mea ns of anti-VSV and anti-mT antibodies and their sorting patterns were analyzed by confocal microscopy, The sorting specificity of MLC isofor ms to sarcomeric sites was shown to increase in the order MLC3nm, to M L1sa, to MLC1sb, to MLC1f and MLC3f following the sequence of developm ental expression, Expressed fast skeletal muscle isoforms (MLC1f and M LC3f) were always localized at the A-bands of myofibrils, while nonmus cle type (MLC3nm) was distributed throughout the cytoplasm, The slow s keletal muscle type (MLC1sa) showed a weak sarcomeric pattern if it wa s co-expressed with MLC3nm, but it was distributed throughout the cyto plasm when expressed in combination with MLC1f, MLC3f or the slow skel etal/ventricular muscle isoform (MLC1sb). The MLC1sb was localized at the A-bands when it was co-expressed with MLC3nm or MLC1sa, while it w as also distributed to the cytoplasm if co-expressed with MLC1f or MLC 3f. Further, expression of chimeric cDNAs revealed that the N-terminal lobe of each isoprotein is responsible for the isoform-specific sorti ng pattern.