Outer mitochondrial membrane cytochrome b 5 (OMb), which is an isoform of cytochrome b 5 (cyt b 5 ) in the endoplasmic reticulum, is a typical tail-anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cyt b 5 that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b 5 , in which the acidic amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem that charged amino acids in the carboxylterminal portion of these proteins determine their locations in the cell.

Cytochrome b5 is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions [Mitoma, J. and Ito, A. (1992) EMBO J. 11, 4197-4203]. In situ topology of cytochrome b5 in the ER-membrane was studied using immunofluorescence microscopy. Antibodies were raised against the hydrophilic portion (anti-b5) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b5 and used for detection of the cytochrome in COS cells which expressed the rat cytochrome. Anti-b5 antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O. The anti-peptide displayed a fluorescence signal only with Triton-permeabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immuno-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O. The results indicate that the carboxy-terminal hydrophilic stretch is exposed to the luminal side. Cytochrome b5 was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method. Anti c-myc monoclonal IgG detected the tagged cytochrome b5 only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane.

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Integration of cytochrome b(5) (b5), a tail-anchored protein located in the endoplasmic reticulum (ER) membrane, into the membrane was studied. Mutation of three amino acids, -Leu-Met-Tyr, at the carboxy-terminal end of the transmembrane segment of b5 to alanines resulted in localization of the mutated protein, b5LMY/AAA, in the cytosol as well as in the ER membrane. When an N-glycosylation site was introduced at the carboxy-terminal end of b5LMY/AAA, a substantial amount of the glycosylated form of the mutant protein was recovered in the cytosol fraction. A portion of the mutant protein recovered in the ER was released from the membrane by incubation with the cytosol fraction, but no further release was observed in the second incubation, suggesting that b5 is present in two different states, loosely-bound and firmly-integrated forms, in the ER membrane. These results suggest that b5 is integrated into the ER membrane via the loosely bound state, in which the carboxy-terminal end of the molecule is inserted into the luminal side of the vesicle but is easily translocated back to the cytosol, and that the three amino acids are important for conversion of the loosely-bound state to the firmly-integrated state.

Solid‐phase methodology has been used to synthesize a series of peptides based on the N‐terminal sequence of human angiotensinogen in which statine (Sta) or the novel analogues (3S, 4S)‐3, 4‐diamino‐ or (3J?, 45)‐3, 4‐diamino‐6‐methylheptanoic acid (Ads or R‐Ads) and (3S, 4S)‐4‐amino‐3‐aminomethyl‐ or (3R, 4S)‐4‐amino‐3‐aminomethyl‐6‐methylheptanoic acid (Amd or R‐Amd) replace either residue 10 or both residues 10–11 at the P1‐P'1 cleavage site. The synthesis of these novel analogues of statine together with biological results on the inhibition of human and rat renin by peptides derived from them is reported. The absolute stereochemistry of the (3S, 4S) Ads was determined by an X‐ray crystallographic analysis of its Nγ‐Boc, Nβ‐Z, R(+)‐1‐methyl benzamide derivative. Peptide Boc‐His‐Pro‐Phe‐His‐Sta‐Val‐Ile‐His‐NH2 (VI) is the best inhibitor of human renin containing Sta at position 10. However, peptides containing Ads and Amd gave better rat renin inhibitors than the corresponding Sta‐containing peptides. Peptide Boc‐His‐Pro‐Phe‐His‐Ads‐Val‐Ile‐His‐NH2 (VII) having Ads at position 10 had an IC50 of 12 nM against rat renin. Although Sta has come to be accepted as an isosteric replacement for a dipeptide unit rather than for a single amino acid residue, in our series of inhibitors Sta is more effective when replacing only the amino acid at position 10 in the natural angiotensinogen sequence. None of the peptides gave any effect in vivo in a hypertensive rat model.

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