Intracellular targeting of isoproteins in muscle cytoarchitecture.

Abstract

Abstract. Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment. IN most cells of higher organisms isoforms, representing closely related proteins with conserved amino acid sequences and often similar functional properties are the products of multigene families. Distinct isoforms may exist as related proteins in different tissues, be developmentally regulated within a single tissue or even coexist within a single cytoplasmic compartment. In part divergence in protein sequence among isoforms may serve to target a given isoprotein to its appropriate compartment within the cell. Specific sequences, often located at the amino terminus, have been found to be responsible for the extracellular export of many secretory proteins (5) or the intracellular targeting of proteins destined for intracellular compartments like mitochondria, chloroplasts or the nucleus (11,17,26).Here we examine the molecular basis for the localization of muscle creatine kinase (MM-CK) ~ within the M-band of the muscle myofibril (50,54,55,56). Creatine kinase is the major enzyme ensuring energy production in the muscle cell. The enzyme is a dimer of two subunits which exist as a nonskeletal muscle form, brain creatine kinase (BB-CK), in embryonic muscle and in nonmuscle cells like brain. Upon muscle differentiation the synthesis of the M-CK subunit is induced and BB-CK is gradually replaced by the skeletal muscle-specific enzyme 14,40,41,51). Although myofibers that differentiate in culture have been shown to contain all three isoenzymes, MM-CK, MB-CK and BB-CK (40, 51) only MM-CK appears to be localized Beat W. Sch~ifer's present address is Department of Pharmacology, Stanford University, Stanford, CA 94305. Abbreviations used in this paper:B-CK, creatine kinase subunit; BB-CK, brain creatine kinase; CMF, calcium-and magnesium-free; MB-CK heterodimeric creatine kinase; M-CK, creatine kinase subunit; MM-CK, muscle creatine kinase. and specifically bound to the M-band of the myofibril (53). This result can be explained in two ways. ...

Intracompartmental sorting of essential myosin light chains: Molecular dissection and in vivo monitoring by epitope tagging

“…Interestingly, attempts to reproduce the sorting of endogenous proteins by introducing labeled proteins, e.g., a-actinins of different sources, into cultured cells (McKenna et al, 1985;Sanger et al, 1986) have hitherto failed. Success was reported, however, by Schafer and Perriard (1988), who showed by microinjecting synthetic RNAs coding for two isoforms of creatine kinase (B-CK and M-CK) and chimeras thereof that the M-CK targeting to the myofibrillar M line appeared to be directed by isoform-specific characteristics embedded mainly in the C-terminal half of the M-CK isoprotein structure. We report in this article, using transfection and microinjection of epitope-tagged LC constructs, that the essential LC is subjected to a precise intracompartmental isoprotein-specific sorting.…”

“…The protein under study from one species has to be introduced into a cellular background devoid of homologous proteins with immune cross-reactivity. This technique allows researchers to follow proteins introduced in the form of a biochemically purified protein, as a synthetic RNA transcribed from full-length cDNA (Schafer and Perriard, 1988), or as cDNA cloned in a eukaryotic expression vector (Bendori et al, 1989;Friedrich et al, 1989;Ngai et al, 1990). The main limitation of this technique is the difficulty of raising antibodies with the necessary specificity.…”

Functional Equivalence of Creatine Kinase Isoforms in Mouse Skeletal Muscle

“…Interestingly there is sequence divergence between MM and BB-CK in the carboxyl-terminal of the enzyme. By expressing M-CK/B-CK fusions it has been shown that the carboxyl terminus of M-CK contains the information necessary for localization to myofibrils (41). In addition, differences in nontranslated regions of CK mRNA have been shown to cause localization of M-CK transcripts to myofibrils and control mRNA stability in B-CK transcripts (42).…”

Phosphorylation of chicken brain‐type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo

“…Therefore, untreated lysates had to be used for cotranslational phosphorylation and incorporation of 32P yielded only very faint signals due to the high endogenous phosphate pools. Incubation was followed by immunoprecipitation [23,27] and extensive washing before preparation of the samples for 2D gel analysis.…”

“…An additional species termed Bb* was also isolated, although its nature remained unresolved [20]. Some minor species identified in cDNA-derived in vitro translation products [22,23], termed Bbl, Bb2 and Bb3 [24], are shown here to be phosphoproteins, which comigrate with Bb*.…”