Abstract
To elucidate the mechanisms of cuproprotein biosynthesis in the secretory pathway, a polyclonal antiserum was generated against hephaestin, a multicopper oxidase essential for enteric iron absorption. Immunoblot analysis and pulse-chase metabolic labeling revealed that hephaestin is synthesized as a single-chain polypeptide modified by N-linked glycosylation to a mature 161-kDa species. Cell surface biotinylation and immunofluorescent studies of polarized, differentiated colon carcinoma cells detected hephaestin on the basolateral surface under steady-state conditions. However, a decrease in the intracellular copper concentration resulted in a marked diminution in the abundance of this protein. Metabolic studies revealed no effect of decreased intracellular copper on the rate of hephaestin synthesis but a dramatic, specific, and reproducible increase in the turnover of the mature 161-kDa protein. Surprisingly, inhibitor studies revealed that this turnover occurs exclusively in the proteasome, and consistent with this finding, in vitro studies identified polyubiquitinated hephaestin under conditions abrogating copper incorporation into this protein. Taken together, these studies demonstrate the presence of a quality control system for posttranslational protein modification occurring beyond the endoplasmic reticulum that, in the case of hephaestin, directly links the rate of enteric iron uptake to nutritional copper status.
Highlights
Copper is an essential transition metal in all aerobic organisms where it functions in specific cuproenzymes to facilitate electron transfer reactions necessary for respiration, antioxidant defense, connective tissue formation, neurotransmitter biosynthesis, peptide amidation, pigment metabolism, and iron homeostasis [1]
Hephaestin synthesized in the presence of tunicamycin was smaller than the products that resulted from endoglycosidase H (Endo H) or peptide N-glycosidase F digestion (Fig. 1C, lanes 4 and 8), suggesting that additional posttranslational modifications occur during biosynthesis
The biosynthetic data indicate that the rate of hephaestin synthesis, folding, and trafficking through the Golgi is unaffected by copper availability and that it is the abundance of the mature glycosylated form of the protein that is altered by the intracellular copper content (Figs. 6 and 7)
Summary
Cell Culture and Antibodies—A full-length hephaestin cDNA was amplified from a human small intestinal cDNA library (Clontech) using nested oligonucleotide primers corresponding to the predicted open reading frame [10] and inserted into pcDNA3.1 (Invitrogen) with a Kozak consensus sequence at the 5Ј end, and the entire nucleotide sequence was verified by automated fluorescent sequencing (PerkinElmer Life Sciences). Cells were pulse-labeled for 20 min with 60 Ci/ml [35S]methionine and [35S]cysteine and chased with regular medium for the indicated time points followed by the collection of lysate for immunoprecipitation and SDS-PAGE as described previously [4]. Limited trypsin proteolysis was performed on hephaestin immunoprecipitated from pulse-labeled T84 cells and incubated in 25 l of PBS containing 2.5–5 ng/l bovine pancreatic trypsin for 1 h at 37 °C. Ubiquitination reaction mixtures contained 40 mM TrisHCl, pH 7.6, 5 mM MgCl2, 1 mM dithiothreitol, 10% glycerol, 1 M ubiquitin aldehyde (Calbiochem), 5 M MG132, protease inhibitor mixture, 1 g/l ubiquitin, 35S-labeled hephaestin, rabbit reticulocyte lysate (Promega), and either an ATP-regenerating system (0.5 mM ATP␥S, 10 mM phosphocreatine, 100 g/ml creatine phosphokinase) or an ATP-depleting system (0.25 g of hexokinase, 10 mM 2-deoxy-Dglucose) as described previously [16]. Reactions were stopped by the addition of Laemmli sample buffer, heated at 72 °C for 15 min, and subjected to 7.5% SDS-PAGE, and proteins were visualized by PhosphorImager (Typhoon 9410, Amersham Biosciences)
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