Preparation of cytochromes b<inf>5</inf> with an extended COOH-terminal hydrophilic segment: Interaction of modified tail-anchored proteins with liposomes in different cholesterol content

Abstract

A group of membrane proteins endowed with a single COOH-terminal hydrophobic domain capable of insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">5</sub> (Hcytb5) having various COOH-terminal extensions with bovine opsin sequence, we constructed expression vectors containing membrane-bound form of Hcytb5 (or Hcytb5op(a), Hcytb5op(b), Hcytb5op(c), and Hcytb5op(d), where NH <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> -terminal bovine opsin sequences with various truncated forms were attached at the COOH-terminus) and established a method for their expression and purification in the holo-form. We analyzed the integration of the TA domain of holo-form of Hcytb5 into protein-free liposomes. The integration of holo- Hcytb5 occurred efficiently into the membranes with a low cholesterol content even in the presence of a small amount of Triton X-100 and, once incorporated, the proteoliposomes were relatively stable.