title: Protein quality--a determinant of the intracellular fate of membrane-bound cytochromes P450 in yeast.
abstract: 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 Saccharomyces cerevisiae and the ultrastructure of the respective transformants was investigated by means of immunoelectron microscopy. As a result, overproduction of both wild-type P450 forms resulted in a massive proliferation of tubular membrane structures distributed over the whole cytoplasm. In contrast, all mutant P450Cm2 and Alk2A forms tested were mainly localized within stacks of paired membranes which often occurred in close vicinity to the nucleus. As found by serial sectioning of a single cell, these stacked membranes bearing the mutant P450 actually represented plates of consecutive membranes arranged one upon the other. A tubular network of endoplasmic reticulum membranes as observed after expression of the wild-type proteins could not be detected. Generally, the kind of mutation introduced into the P450 forms did not influence the morphology of the induced membranes. Even single amino acid exchanges in the cytosolic domain caused the formation of membrane stacks. The common feature of all mutant P450 forms causing the formation of stacked membranes was, however, their lower protein stability after heterologous expression in the S. cerevisiae host cells, compared to the stability of the authentic cytochromes P450. Furthermore, the proliferated membranes containing the different P450 forms were characterized by means of subcellular fractionation experiments. Using this approach, clear differences in the distribution of spectrally active and inactive P450 molecules were found. The results obtained suggest the presence of an intracellular sorting mechanism based on the protein quality, which finally leads to the differences in the intracellular distribution of wild-type and mutant cytochromes P450.
authors.affiliation: Max Delbrück Center for Molecular Medicine, Berlin-Buch, Federal Republic of Germany.
authors.forename: T
authors.initials: T
authors.lastname: Zimmer
authors.forename: F
authors.initials: F
authors.lastname: Vogel
authors.forename: A
authors.initials: A
authors.lastname: Ohta
authors.forename: M
authors.initials: M
authors.lastname: Takagi
authors.forename: W H
authors.initials: WH
authors.lastname: Schunck
mesh_terms: D002175:Candida / Q000235:genetics*; D002451:Cell Compartmentation; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D007425:Intracellular Membranes / Q000648:ultrastructure; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000648:ultrastructure
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System