Subunit assembly and secretion of transthyretin: studies in a cell-free translation system and in microinjected Xenopus oocytes.
Transthyretin (TTR), or thyroid-binding prealbumin, is a protein of 55 kDa, composed of four identical subunits, which is synthesized by the liver and choroid plexus epithelium. In order to study the subunit assembly and secretion of TTR, cRNA encoding TTR was translated in a rabbit reticulocyte lysate or microinjected into Xenopus oocytes, and radiolabelled biosynthetic products were immunoprecipitated with an antibody against TTR and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. In the cell-free translation system in the absence of dog pancreatic microsomes, a single protein of Mr 17,000 was synthesized. In the presence of dog pancreatic microsomes, two proteins of Mr 15,000 and 37,000 were observed. The Mr 17,000 protein was identified as pre-TTR and the Mr 15,000 and 37,000 proteins as monomeric and dimeric forms of TTR. When the mRNA was microinjected into Xenopus oocytes both Mr 15,000 and 37,000 proteins were secreted into the media. It was shown that, under the SDS-PAGE conditions used in this study, the TTR tetramer dissociated to the dimeric form (Mr 37,000), but that there was no, or at least very little, further breakdown to the monomer. Therefore, to determine whether tetrameric or dimeric forms of TTR were secreted from the oocytes, the media from microinjected oocytes were subjected to gel permeation chromatography under non-dissociating conditions, and the eluted fractions analysed by SDS-PAGE. TTR eluted from the column as a dimer; there was no tetramer or monomer. The dimer, however, was completely dissociated to the monomer when analysed by SDS-PAGE, which suggested that incomplete or incorrect subunit assembly had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)
Docherty
K
K
Department of Medicine, University of Birmingham, Queen Elizabeth Hospital.
Shennan
K I
KI
Marsden
R F
RF
Ramsden
D B
DB
D000818:Animals; D002474:Cell-Free System; D002850:Chromatography, Gel; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008845:Microinjections; D008861:Microsomes / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D011228:Prealbumin / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics; D018075:RNA, Complementary; D011817:Rabbits; D014982:Xenopus laevis
D011228:Prealbumin; D018075:RNA, Complementary; D012313:RNA