Biosynthetic precursors and in vitro translation products of the glucose transporter of human hepatocarcinoma cells, human fibroblasts, and murine preadipocytes

Abstract

Antisera to the human erythrocyte Glc transporter immunoblotted a polypeptide of Mr 55,000 in membranes from human hepatocarcinoma cells, Hep G2, human fibroblasts, W138, and murine preadipocytes, 3T3-L1. This antisera immunoprecipitated the erythrocyte protein which had been photoaffinity labeled with [3H]cytochalasin B, immunoblotted its tryptic fragment of Mr 19,000, and immunoblotted the deglycosylated protein as a doublet of Mr 46,000 and 38,000. This doublet reduced to a single polypeptide of Mr 38,000 after boiling. When Hep G2, W138, and 3T3-L1 cells were metabolically labeled with L-[35S]methionine for 6 h, a broad band of Mr 55,000 was immunoprecipitated from membrane extracts. In pulse-chase experiments, two bands of Mr 49,000 and 42,000 were identified as putative precursors of the mature transporter. The t1/2 for mature Glc transporter was 90 min for Hep G2 cells that had been starved for methionine (2 h) and pulsed for 15 min with L-[35S]methionine. Polypeptides of Mr 46,000 and 38,000 were immunoprecipitated from Hep G2 cells that had been metabolically labeled with L-[35S]methionine in the presence of tunicamycin. This doublet reduced to the single polypeptide of Mr 38,000 after boiling. In the absence of tunicamycin, but not in its presence, mature polypeptide of Mr 55,000 was immunoprecipitated from Hep G2 cells metabolically labeled with D-[3H]GlcN. A polypeptide of Mr 38,000 was observed in boiled immune complexes from the in vitro translation products of Hep G2, W138, and 3T3-L1 cell RNA. Dog pancreatic microsomes cotranslationally, but not posttranslationally, converted this to a polypeptide of Mr 35,000. A model for Glc transporter biogenesis is proposed in which the primary translation product of Mr 38,000 is converted by glycosylations to a polypeptide of Mr 42,000. The latter is then processed via heterogeneous complex N-linked glycosylations to form the mature Glc transporter, Mr 55,000.