Characterization of the subunit structure of the catalytically active type I iodothyronine deiodinase.
Type I iodothyronine deiodinase is a approximately 50-kDa, integral membrane protein that catalyzes the outer ring deiodination of thyroxine. Despite the identification and cloning of a 27-kDa selenoprotein with the catalytic properties of the type I enzyme, the composition and the physical nature of the active deiodinase are unknown. In this report, we use a molecular approach to determine holoenzyme composition, the role of the membrane anchor on enzyme assembly, and the contribution of individual 27-kDa subunits to catalysis. Overexpression of an immunologically unique rat 27-kDa protein in LLC-PK1 cells that contain abundant catalytically active 27-kDa selenoprotein decreased deiodination by approximately 50%, and > 95% of the LLC-PK1 derived 27-kDa selenoprotein was specifically immune precipitated by the anti-rat enzyme antibody. The hybrid enzyme had a molecular mass of 54 kDa and an s(20,w) of approximately 3.5 S indicating that every native 27-kDa selenoprotein partnered with an inert rat 27-kDa subunit in a homodimer. Enzyme assembly did not depend on the presence of the N-terminal membrane anchor of the 27-kDa subunit. Direct visualization of the deiodinase dimer showed that the holoenzyme was sorted to the basolateral plasma membrane of the renal epithelial cell.
Leonard
J L
JL
Department of Cellular and Molecular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. jack leonard@UMASSMED.edu
Visser
T J
TJ
Leonard
D M
DM
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D019281:Dimerization; D004847:Epithelial Cells / Q000201:enzymology; D020035:Holoenzymes; D007453:Iodide Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012643:Selenium; D013045:Species Specificity; D013552:Swine
D020035:Holoenzymes; D008565:Membrane Proteins; D008667:Metalloproteins; D011994:Recombinant Proteins; C109812:iodothyronine deiodinase type I; D007453:Iodide Peroxidase; D012643:Selenium