Secretion and lipid association of human apolipoprotein E in Saccharomyces cerevisiae requires a host mutation in sterol esterification and uptake.
We have expressed a cDNA to human apolipoprotein E (apoE) in Saccharomyces cerevisiae. Secretion of apoE was achieved only by the use of a mutant (upc2) strain of yeast with the phenotype of enhanced uptake and intracellular esterification of exogenous cholesterol. Approximately 40 ng/ml apoE was secreted by upc2 mutants in the absence of media cholesterol. ApoE secretion was increased 2-3-fold upon the inclusion of cholesterol in the growth media. This response to exogenous cholesterol was not mediated at the transcriptional level, since apoE mRNA levels were constant under all culture conditions. Concomitant with the increase in secretion following cholesterol uptake by upc2 strains, approximately 5% of secreted apoE was associated with lipid; polar and non-polar lipids were detected in this lipoprotein fraction. Intracellular degradation of apoE in non-secreting strains of yeast was minimized by the presence of null mutations in both vacuolar proteases with non-specific activity (pep4) and a Golgi endopeptidase with specificity for paired basic residues (kex2). The approach of expressing human apolipoproteins in yeast may identify factors that mediate lipoprotein biosynthesis in higher cells. One such factor could be the mammalian equivalent of the gene product of UPC2.
Sturley
S L
SL
Department of Biochemistry, University of Wisconsin, Madison 53706.
Culbertson
M R
MR
Attie
A D
AD
D001057:Apolipoproteins E / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D004951:Esterification; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D015151:Immunoblotting; D050356:Lipid Metabolism; D009154:Mutation; D043484:Proprotein Convertases; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins
D001057:Apolipoproteins E; D029701:Saccharomyces cerevisiae Proteins; D002784:Cholesterol; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae