Abstract
We have examined the possible role of clathrin-coated vesicles (CVs) in the genesis of the sarcoplasmic reticulum (SR) in developing chick skeletal myotubes. Calsequestrin (CSQ) a luminal Ca2+ binding protein of the terminal SR cisternae, is contained within the vesicle lumen of skeletal muscle CVs in substantial amounts, approximately four molecules/CV. Employing 3-day cultures of chick skeletal myotubes we demonstrate that after a 30-min labeling with [35S]methionine and cysteine, radioactivity in CSQ remains high in the CVs 45 min later and then declines, while labeled CSQ in the SR continues to rise. No CSQ appears to be secreted. All of the CSQ in both the CVs and SR is sensitive to the activity of endoglycosidase H, and a significant fraction also binds to wheat germ agglutinin. Based on these results, we discuss the hypothesis that a selective CV-mediated pathway exists in developing skeletal muscle cells for the transport of CSQ from the early/intermediate Golgi apparatus to the SR.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Acetylcholinesterase / metabolism
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Acetylglucosaminidase / metabolism
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Animals
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Biological Transport
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Blotting, Western
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Calcium-Transporting ATPases / metabolism
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Calsequestrin / metabolism*
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Cells, Cultured
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Centrifugation, Density Gradient
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Chick Embryo
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Clathrin / metabolism*
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Coated Pits, Cell-Membrane / metabolism*
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Cysteine / metabolism
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Electrophoresis, Agar Gel
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Endosomes / metabolism*
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Golgi Apparatus / metabolism*
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Immunosorbent Techniques
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Kinetics
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Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
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Methionine / metabolism
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Muscle Proteins / metabolism*
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Muscles / embryology
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Muscles / metabolism
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Muscles / ultrastructure
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Sarcoplasmic Reticulum / metabolism*
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Wheat Germ Agglutinins / metabolism
Substances
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Calsequestrin
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Clathrin
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Muscle Proteins
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Wheat Germ Agglutinins
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Methionine
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Acetylcholinesterase
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Acetylglucosaminidase
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Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
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Calcium-Transporting ATPases
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Cysteine