Biochemical characterization and intracellular localization of the Menkes disease protein

Abstract

Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells. Pulse-chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form. Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast.

Figure 1

(A) RNA blot analysis of Menkes mRNA in cell lines from Menkes patients (lanes 1–3), control primary fibroblasts (lanes 4 and 5), and human cell lines (lanes 6–9). Ten micrograms of total RNA was isolated, electrophoresed, transferred to nylon, and hybridized with a human Menkes [³²P]cRNA probe. (B) Immunoblot of cell lysates with Menkes (lanes 1–5) or Wilson (lanes 6–10) antibody. Protein was loaded at 75 μg per lane, and after SDS/10% PAGE proteins were transferred to nitrocellulose and analyzed by ECL (Amersham).

Figure 2

Immunoprecipitation of Menkes ATPase from HeLa cell lysates. Following a 1-h pulse-label with [³⁵S]methionine and [³⁵S]cysteine, cells were lysed and immunoprecipitated with preimmune sera (lane 1), Menkes antibody (lanes 2, 4, and 6–9), or Menkes antibody preincubated with Menkes fusion protein (lanes 3 and 5). For some experiments cells were pretreated with tunicamycin (4 μg/ml) for 2 h prior to immunoprecipitation (lanes 4, 5, and 9). Immunoprecipitated Menkes protein was digested with endo H or endo F (lanes 7 and 8) as described. Samples were electrophoresed on SDS/5-12% polyacrylamide gradient gels and subjected to fluorography.

Figure 3

(A) Pulse–chase labeling of biosynthesized Menkes ATPase in HeLa cells. Cultures were pulsed for 10 min with [³⁵S]methionine and [³⁵S]cysteine at 500 μCi/ml and then chased for indicated times in media containing excess methionine and cysteine. Immunoprecipitates of cell lysates were analyzed by SDS/10% PAGE and fluorography. (B) HeLa cells were pulse-labeled for 10 min (lanes 1–3) followed by a 1-h chase (lanes 4–6). Cell lysates were immunoprecipitated with Menkes antibody and incubated without enzyme (lanes 1 and 4) or digested with endo H (lanes 2 and 5) or endo F (lanes 3 and 6).

Figure 4

Immunoblot analysis of HeLa cell lysates following isopycnic centrifugation in a continuous linear sucrose gradient. A 75-μg sample of protein from each fraction was separated by SDS/10% PAGE, transferred to nitrocellulose, and immunoblotted with Menkes antibody or antibodies to TAP-1, γ-adaptin (AP-1), or the cation-independent mannose-6-phosphate receptor (MPR).

Figure 5

Confocal immunofluorescence localization of the Menkes ATPase in HeLa cells. Cells were processed for indirect immunofluorescence and analyzed after incubation with antibodies to the Menkes ATPase (a, d, g, and j) and γ-adaptin (AP-1) (b), the cation-independent mannose-6-phosphate receptor (MPR) (e), lamp-1 (h), and AP-2 (k). The combined confocal image from each double-labeling is shown in c, f, i, and l. (×400.)