A model system for studying membrane biogenesis: overexpression of cytochrome B5 in yeast results in marked proliferation of the intracellular membrane*
Authors: Vergères Guy , Yen T. S. Benedict , Aggeler Judith , Lausier James , Waskell Lucy 
Published: April 2021

Cytochrome b5 is an amphipathic microsomal protein that is anchored to the endoplasmic reticulum by a single hydrophobic transmembrane α-helix located near the carboxyl terminus of the protein. In yeast, cytochrome b5 provides electrons for fatty acid desaturation and ergosterol biosynthesis. High level expression of cytochrome b5 in Saccharomyces cerevisiae was achieved using the yeast metallothionein promoter and a synthetic cytochrome b5 gene.

In order to accommodate the markedly increased amount of the membrane-bound cytochrome b5, the yeast cell proliferated its nuclear membrane. As many as 20 pairs of stacked membranes could be observed to partially encircle the nucleus. This morphological arrangement of membrane around the nucleus is known as a karmella. In an effort to understand which part of the cytochrome b5 molecule, i.e. the membrane anchor or the soluble heme domain, which is competent in electron transfer, provided the signal for the de novo membrane biogenesis, a series of studies, including site- directed mutagenesis, was undertaken. The results of these experiments demonstrated that the inactive hemedeficient apo form of the membrane-bound protein stimulates membrane proliferation to the same extent as the holo wild-type protein, whereas cytosolic forms of cytochrome b5 did not induce membrane synthesis. These data demonstrate that membrane proliferation is a consequence of the cell’s ability to monitor the level of membrane proteins and to compensate for alterations in these levels rather than the result of the ability of the extra cytochrome b5 to catalyze synthesis of extra lipid that had to be accommodated in new membrane.

Site-directed mutagenesis studies of the membrane binding domain of cytochrome b5 provided additional clues about the nature of the signal for membrane proliferation. Replacement of the membrane anchor by a non-physiological nonsense sequence of 22 leucines gave rise to a mutant protein that triggered membrane biosynthesis. The conclusion from these experiments is clear; the signal for membrane proliferation does not reside in some specific amino acid sequence but instead in the hydrophobic properties of the proliferant. Interestingly, these membranes are somewhat diminished in quantity and have a slightly altered morphology compared to those induced by the wild-type protein. It was also observed that disruption of the putative a helix of the membrane anchor by an Ala116Pro mutation, which gives rise to two sequential prolines at positions 115 and 116 results in a protein with diminished capacity to induce membrane formation. This finding suggests that the secondary structure of the membrane binding domain may be an important component of the signal for membrane biosynthesis. In summary, cytochrome b5, with its single-helical transmembrane anchor is an excellent model system in which to investigate the molecular mechanism whereby the cell regulates membrane biogenesis.