A nontetrameric species is the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte lysates.
Inside the interphase cell, approximately 5% of the total intermediate filament protein exists in a soluble form. Past studies using velocity gradient sedimentation (VGS) indicate that soluble intermediate filament protein exists as an approximately 7 S tetrameric species. While studying intermediate filament assembly dynamics in the Xenopus oocyte, we used both VGS and size-exclusion chromatography (SEC) to analyze the soluble form of keratin. Previous studies (Coulombe, P. A., and E. Fuchs. 1990. J. Cell Biol. 111:153) report that tetrameric keratins migrate on SEC with an apparent molecular weight of approximately 150,000; the major soluble form of keratin in the oocyte, in contrast, migrates with an apparent molecular weight of approximately 750,000. During oocyte maturation, the keratin system disassembles into a soluble form (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787) and the amount of the 750-kD keratin complex increases dramatically. Immunoprecipitation analysis of soluble keratin from matured oocytes revealed the presence of type I and type II keratins, but no other stoichiometrically associated polypeptides, suggesting that the 750-kD keratin complex is composed solely of keratin. To further study the formation of the 750-kD keratin complex, we used rabbit reticulocyte lysates (RRL). The 750-kD keratin complex was formed in RRLs contranslating type I and type II Xenopus keratins, but not when lysates translated type I or type II keratin RNAs alone. The 750-kD keratin complex could be formed posttranslationally in an ATP-independent manner when type I and type II keratin translation reactions were mixed. Under conditions of prolonged incubation, such as occur during VGS analysis, the 750-kD keratin complex disassembled into a 7 S (by VGS), 150-kD (by SEC) form. In urea denaturation studies, the 7 S/150-kD form could be further disassembled into an 80-kD species that consists of cofractionating dimeric and monomeric keratin. Based on these results, the 750-kD species appears to be a supratetrameric complex of keratins and is the major, soluble form of keratin in both prophase and M-phase oocytes, and RRL reactions.
Bachant
J B
JB
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309-0347, USA.
Klymkowsky
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D007382:Intermediate Filaments / Q000502:physiology*; D007633:Keratins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012995:Solubility; D014508:Urea / Q000494:pharmacology; D014981:Xenopus
D012333:RNA, Messenger; D007633:Keratins; D014508:Urea