In vivo biosynthesis of clathrin and other coated vesicle proteins from rat liver.
A biosynthetic study of rat liver coated vesicle (CV) proteins was undertaken by using in vivo labeling with L-[35S]methionine. CVs were isolated and purified by using standard procedures and characterized by electron microscopy, sedimentation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography, or by gel slicing and liquid scintillation counting. After 5 1/2 min of labeling (the earliest time examined), incorporation of radioactive clathrin heavy-chain (180-kD (kilodalton] subunits as well as a 90-kD CV-associated protein into purified CVs was demonstrated. The level of labeled 180-kD clathrin in coated vesicles increased rapidly during the first 2 hr of labeling and then continued to rise at a slower rate between 4 and 16 hr. This slow accumulation of labeled clathrin heavy chains in the CV pool may reflect early compartmental sequestration of a fraction of newly synthesized clathrin with delayed assembly into free CVs. By 16 hr of labeling, clathrin 180-kD chains and the 90-kD CV-associated protein accounted for approximately 48 and 26%, respectively, of the radioactivity in all CV proteins. Two proteins of MWa 68 kD and 53 kD showed marked declines in cpm/unit protein between 30 min and 4 hr, raising the possibility that these species may be transferred out of CVs during or after transport without loss of the other CV proteins. The possibility is also raised that clathrin heavy chains may be recycled during CV formation. Possible heterogeneity within individual CV preparations with respect to protein composition and derivation from both plasma membrane and Golgi regions are proposed.
Pierce
L R
LR
Zurzolo
C
C
Edelhoch
H
H
D000818:Animals; D002499:Centrifugation, Density Gradient; D002966:Clathrin / Q000096:biosynthesis*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors
D002966:Clathrin; D008715:Methionine