Abstract
The rat S14 gene encodes a protein of unknown function and has an amino acid sequence unrelated to any published sequences. Expression of mRNA S14 and lipogenesis in liver, fat, and mammary gland are regulated coordinately by dietary and hormonal stimuli, suggesting that the S14 protein may be associated with lipogenesis. Antisera to synthetic peptides corresponding to portions of the deduced amino acid sequence of the protein were used to identify the protein and to compare its regulation with that of mRNA S14. Antisera specifically recognized the in vitro translation product of mRNA S14 as defined by its migration on two-dimensional gel electrophoresis. A product of identical Mr was identified on Western blots of liver homogenates from hyperthyroid, carbohydrate-fed rats. Subcellular fractionation showed that S14 protein is primarily cytosolic. The protein was detectable in tissues with abundant S14 gene expression, including hyperthyroid liver and epididymal fat and hypothyroid brown adipose tissue, whereas it was undetectable in hypothyroid liver and euthyroid kidney, testis, and spleen. Diurnal variation in hepatic mRNA S14 correlated with comparable changes in levels of the protein. Surprisingly, no S14 protein was observed in the livers of chronically (3 week) hypothyroid rats treated with triiodothyronine (T3) until 12 h had elapsed, despite attainment of maximal levels of mRNA S14 within 4 h. Rapid appearance of protein after T3 treatment was observed in both euthyroid and short term (4 day) hypothyroid rats, suggesting that long-term hypothyroidism is associated with a defect in the translational efficiency of mRNA S14.
Highlights
Repeated searches of the existing sequence repositories have revealed no sequences related to thededuced 5314 amino acid sequence published by Liaw and Towle (8).In order to study the S14 protein, we raised antisera tosynthetic peptides rats
Diurnal variation in hepaticmRNA S14 correlated with comparable changes in levels of Synthesis of Antigen and Immunization-The S14 cDNA nucleotide sequence(8) predicted a150-residuepeptide.Hydrophilicity the protein
Rapid appearanceof protein after T 3 treatment was observed in both euthyroid and short term (4 day) hypothyroid rats, suggesting that longterm hypothyroidism is associated with a defectin the translational efficiency of mRNA 514
Summary
Repeated searches of the existing sequence repositories have revealed no sequences related to thededuced 5314 amino acid sequence published by Liaw and Towle (8).In order to study the S14 protein, we raised antisera tosynthetic peptides rats. Antipeptide (81-117) sera specificallyrecognized the in vitro translation product (Mr19,000,PI 4.9) originally designated as “spot 14” (15).Total [35S]methionine-labeledtranslation products of hyperthyroid rat hepatic mRNA (2 X lo5 trichloroacetic acid-insoluble counts/min) were incubated with antiserum with or without excess (20 pg) peptide Immunoprecipitated protein was separated by two-dimensional electrophoresis (Fig. 1).The precipitated translational produce exhibited the M , (19,000) and acidic PI (4.9) characteristic of “spot 14” and was quantitatively displaced from the precipitate by incubation with excess peptide (81117).
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