title: Cell-free incorporation of newly synthesized myosin subunits into thick myofilaments.
abstract: Although a substantial literature exists on the in vitro polymerization of purified myosin, little is known about native thick filament assembly, remodeling or turnover. We have recently described a cell-free system (Bouche et al., 1988) to examine the interactions between thick filaments and soluble, newly synthesized myofibrillar proteins. In the present manuscript we describe our studies on myosin heavy (MHC) and light chain (LC) incorporation into myofibrils or native and synthetic thick filaments. 35S-labeled myofibrillar proteins or myosin subunits were synthesized in a reticulocyte lysate translation system after which myofibrils or myofilaments were added and incubated with these proteins in the lysate. The added filaments were then sedimented and analyzed by SDS-PAGE and fluorography to establish which of the labeled protein subunits were co-pelleted. Operationally, this co-sedimentation of labeled proteins with myofilaments has been termed 'protein incorporation'. We observed that newly synthesized MHC, LCs 1, 2 and 3 all incorporated into the thick filaments. However, the quantity and specificity of LC incorporation depended upon the structure or composition of the filaments. LCs 1 and 3 were preferentially incorporated into myofibrils and native thick filaments, whereas LC2 was selectively taken up by synthetic filaments prepared from purified myosin. These results suggest that soluble MHCs and LCs interact independently with myofilaments. This hypothesis is supported by the observation that selective removal of soluble MHCs, or of a single LC, did not alter the incorporation of the remaining myosin subunits. Similarly, MHCs synthesized in the absence of LCs also incorporated into myofilaments or myofibrils. We propose that myosin subunits are capable of independent incorporation into and exchange from myofilaments.
authors.affiliation: Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021.
authors.forename: S M
authors.initials: SM
authors.lastname: Goldfine
authors.forename: S
authors.initials: S
authors.lastname: Einheber
authors.forename: D A
authors.initials: DA
authors.lastname: Fischman
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins; D002474:Cell-Free System; D002642:Chick Embryo; D009124:Muscle Proteins / Q000378:metabolism; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D014336:Troponin / Q000378:metabolism; D019209:Troponin C; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D009124:Muscle Proteins; D009218:Myosins; D012333:RNA, Messenger; D014336:Troponin; D019209:Troponin C; D000099163:Myosin Binding Protein C