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The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ∼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.

Nucleotide substitutions in the gene for NADH-cytochrome b5 reductase were identified in three independent probands of hereditary methemoglobinemia type I. Patients in Kagoshima and Okinawa in Japan were shown to possess the same base change, from guanine to adenine at codon 57, which results in amino acid substitution from Arg to Gln. This nucleotide change was the same as formerly found in a patient in Toyoake, Japan (Katsube, T., Sakamoto, N., Kobayashi, Y., Seki, R., Hirano, M., Tanishima, K., Tomoda, A., Takazakura, E., Yubisui, T., Takeshita, M., Sakaki, Y., and Fukumaki, Y. (1991) Am. J. Hum. Genet. 48, 799-808). A type I patient in Italy was shown to have a base change from guanine to adenine at codon 105 which causes substitution from Val to Met. To characterize the enzymes of type I patients, Arg-57----Gln and Val-105----Met mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity. kcat/Km values (NADH) of these two enzymes were 25% in Arg-57----Gln and 14.5% in Val-105----Met compared with that of the wild type enzyme, while the value of type II (generalized, severe form of the disease) mutant enzyme was 3% of the normal value (Yubisui, T., Shirabe, K., Takeshita, M., Kobayashi, Y., Fukumaki, Y., Sakaki, Y., and Takano, T. (1991) J. Biol. Chem. 266, 66-70). The type I mutant enzymes were less heat-stable and more susceptible to proteinase treatment than the wild type. From these results we conclude that restriction of enzyme deficiency to red cells in hereditary methemoglobinemia type I may be generally derived from instability and increased proteolytic susceptibility of variant NADH-cytochrome b5 reductases due to a point mutation.

The subcellular distribution of rat erythrocyte NADH-cytochrome b5 reductase was determined by radioimmunoassay, using a rabbit antibody against the cathepsin D cleaved water-soluble fragment of rat liver microsomal reductase (I-reductase), which is known to be immunologically similar to the red cell enzyme. Erythrocytes contained approximately 30 ng of reductase/mg of protein, of which 90% were recovered in the hemolysate supernatant and 2.3% in the ghost fraction. After concentration by precipitation with 70% saturated (NH4)2SO4, the NADH-cytochrome c reductase activity of the soluble enzyme could be assayed in the presence of cytochrome b5, and was found to be inhibited by anti 1-reductase antibodies. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobilities of erythrocyte membrane-associated and soluble reductase of the liver microsomal enzyme and its cathepsin D cleaved hydrophilic fragment (I-reductase) were examined in crude fractions by blotting followed by specific and highly sensitive immunostaining. The intact microsomal enzyme and the two erythrocyte reductases all had similar mobilities and migrated behind 1-reductase. However, the ghost-associated reductase, which was not attributable to contaminating leukocyte or reticulocyte membranes, was distinguishable from the soluble form by two criteria: (i) a lower dependence on exogenous cytochrome b5 in the NADH-cytochrome c reductase assay; and (ii) a larger apparent Mr upon gel filtration in the presence of Triton X-100, presumably because of detergent binding. Considering these results, possible biogenetic relations between membrane-bound and soluble erythrocyte reductase are discussed.