Molecular cloning and characterization of two isoforms of Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase.

Esterification of cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT) is a key element in maintaining cholesterol homeostasis in cells of higher animals. In the budding yeast, Saccharomyces cerevisiae, accumulation of ergosteryl esters accompanies entry into stationary phase and sporulation. We have determined that two genes in yeast, SAT1 and SAT2, encode isozymes of acyl-CoA:sterol acyltransferase (ASAT) which are functionally related to ACAT. The SAT1 isozyme is the major catalytic isoform, accounting for at least 65-75% of total ASAT activity. Targeted deletions of one or both genes do not compromise mitotic cell growth or spore germination. However, diploids that are homozygous for a SAT1 null mutation exhibit significantly reduced sporulation efficiency. Furthermore, a larger fraction of the sporulating diploids arrest after the first meiotic division. Human ACAT expressed in sat1 sat2 mutant cells can catalyze esterification of cholesterol and, to a lesser extent, ergosterol in vitro, but restores ergosteryl oleate formation in vivo to only approximately 8% of that catalyzed by yeast ASAT in wild-type cells.

Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast.

Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SEC61 (Deshaies, R. J., and R. Schekman. 1987. J. Cell Biol. 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.

Secretion in yeast: structural features influencing the post-translational translocation of prepro-alpha-factor in vitro.

In vitro, efficient translocation and glycosylation of the precursor of yeast alpha-factor can take place post-translationally. This property of prepro-alpha-factor appears to be unique as it could not be extended to other yeast protein precursors such as preinvertase or preprocarboxypeptidase Y. In order to determine if specific domains of prepro-alpha-factor were involved in post-translational translocation, we carried out a series of experiments in which major domains were either deleted or fused onto reporter proteins. Fusion of various domains of prepro-alpha-factor onto the reporter protein alpha-globin did not allow post-translational translocation to occur in the yeast in vitro system. Prepro-alpha-factor retained its ability to be post-translationally translocated when parts or all of the pro region were deleted. Removal of the C-terminal repeats containing mature alpha-factor had the most profound influence as post-translational translocation decreased in proportion to the number of repeats deleted. Taken together, these results suggest that efficient post-translational translocation requires a signal sequence and the four C-terminal repeats. There does not however, appear to be specific information contained within the C-terminus, as their presence in fusion did not enable the post-translational translocation of reporter proteins. Lastly, the ability to post-translationally translocate radiochemically pure prepro-alpha-factor that had been isolated by immuno-affinity chromatography required the addition of a yeast lysate fraction. Moreover, post-translational translocation is a function of the microsomal membrane of yeast microsomes and not of a factor peculiar to the yeast lysate, as reticulocyte lysate supported this as well.

Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism.

In an in vitro system comprising a yeast cell-free translation system, yeast microsomes and mRNA encoding prepro-alpha-factor, the translocation of this protein across the membrane of the microsomal vesicle and its glycosylation could b uncoupled from its translation. Such post-translational processing is dependent upon the presence of ATP in the system. It is not, however, affected by a variety of uncouplers, ionophores or inhibitors, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), valinomycin, nigericin, dinitrophenol (DNP), potassium cyanide (KCN) or N-ethyl maleimide (NEM). This mechanism of translocation is significant as it indicates that a protein of 18 000 daltons is capable of crossing an endoplasmic reticulum-derived membrane post-translationally. For the moment, this phenomenon seems to be restricted to prepro-alpha-factor in the yeast in vitro system. Neither invertase nor IgG chi light chain could be translocated post-translationally in yeast, nor was such processing observed for prepro-alpha-factor in a wheat germ system supplemented with canine pancreatic microsomes.

Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system.

A homologous cell-free system has been derived from the yeast Saccharomyces cerevisiae that allows the translation, translocation, and glycosylation of the precursors of yeast mating factor alpha and invertase. The precursors were translated in a yeast lysate from mRNA obtained by in vitro transcription of the MF alpha 1 and SUC2 genes. Inclusion of yeast microsomes resulted in the glycosylation of the alpha-factor precursor, which was demonstrated to be sequestered within the membrane vesicles. Similar results, including signal sequence cleavage, were observed for invertase. Processing of secretory proteins translated in a yeast lysate could not be achieved using microsomes derived from canine pancreas, nor were yeast microsomes active in a wheat germ translation system.