THE N-TERMINAL HYDROPHOBIC DOMAIN OF P450C21 IS REQUIRED FOR MEMBRANEINSERTION AND ENZYME STABILITY

Microsomal cytochromes P-450 are known to be integrated into smooth en doplasmic reticulum through their hydrophobic sequences located at the N termini. The length requirement of the membrane insertion signal wa s determined by the generation of six plasmids encoding mutant P450c21 that lacked various portions of the N-terminal hydrophobic domains. W hen they were transcribed and translated in vitro in the presence of e ndoplasmic reticulum membranes, mutant protein lacking more than a thi rd of the first hydrophobic domain gradually lost the ability to inser t into the membrane and stayed mostly in the soluble fraction when the first N-terminal hydrophobic domain was removed. The steady-state amo unt of the truncated proteins was progressively reduced in parallel to the extent of their N-terminal deletions, due to their fast degradati on. This process was accompanied by a decrease in the enzymatic activi ty. Therefore, the first hydrophobic domain of P450c21 not only serves as a membrane targeting and anchoring domain, but it is also importan t for the in vivo protein stability.