PROTEIN-QUALITY - A DETERMINANT OF THE INTRACELLULAR FATE OF MEMBRANE-BOUND CYTOCHROMES P450 IN YEAST

To elucidate mechanisms determining the intracellular localization of cytochromes P450, authentic and mutant cytochromes P450 52A4 (P450Cm2) and P450 52A5 (P450Alk2A) were heterologously expressed in Saccharomy ces cerevisiae and the ultrastructure of the respective transformants was investigated by means of immunoelectron microscopy. As a result, o verproduction of both wild-type P450 forms resulted in a massive proli feration of tubular membrane structures distributed over the whole cyt oplasm. In contrast, all mutant P450Cm2 and Alk2A forms tested were ma inly localized within stacks of paired membranes which often occurred in close vicinity to the nucleus. As found by serial sectioning of a s ingle cell, these stacked membranes bearing the mutant P450 actually r epresented plates of consecutive membranes arranged one upon the other . A tubular network of endoplasmic reticulum membranes as observed aft er expression of the wild-type proteins could not be detected, General ly, the kind of mutation introduced into the P450 forms did not influe nce the morphology of the induced membranes, Even single amino acid ex changes in the cytosolic domain caused the formation of membrane stack s. The common feature of all mutant P450 forms causing the formation o f stacked membranes was, however, their lower protein stability after heterologous expression in the S. cerevisiae host cells, compared to t he stability of the authentic cytochromes P450. Furthermore, the proli ferated membranes containing the different P450 forms were characteriz ed by means of subcellular fractionation experiments. Using this appro ach, clear differences in the distribution of spectrally active and in active P450 molecules were found. The results obtained suggest the pre sence of an intracellular sorting mechanism based on the protein quali ty, which finally leads to the differences in the intracellular distri bution of wild-type and mutant cytochromes P450.