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Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoBcontaining lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 M), but resistant to N-acetyl-leucylleucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7␣-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7␣-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.The assembly and secretion of lipoproteins containing apoB occur in several tissues (liver, intestine, yolk sac, and heart) and are essential for the transport and delivery of fat during development and in the adult (reviewed in Refs. 1-4). There are at least three distinct human familial disorders associated with developmental and nutritional abnormalities caused by the inability of the liver and/or intestine to assemble and secrete apoB-containing lipoproteins (5). Hypobetalipoproteinemia is caused by mutations of the apoB gene leading to forms that are incapable of forming lipoprotein particles and/or to impaired synthesis of apoB by the liver and intestine (1, 6). The expression of human hypobetalipoproteinemic apoB genes in mice recapitulates many of the phenotypic abnormalities observed in human (7,8). Abetalipoproteinemia is caused by mutations in the MTP 1 gene (9 -12). The absence of MTP lipid transfer activity in the lumen of the endoplasmic reticulum (ER) causes apoB to be degraded rather than secreted as a lipoprotein particle by the liver and intestine (12, 13). The recent demonstration that inactivation of a single allele of the MTP gene in mice causes signifi...

Phosphomevalonate kinase catalyzes the conversion of mevalonate-5-phosphate to mevalonate-5-diphosphate and was originally believed to be a cytosolic enzyme. In this study we have localized the phosphomevalonate kinase gene to chromosome 1p13-1q22-23 and present a genomic map indicating that the gene spans more than 8.4 kb in the human genome. Furthermore, we show that message levels and enzyme activity of rat liver phosphomevalonate kinase are regulated in response to dietary sterol levels and that this regulation is coordinate with 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol biosynthesis. In addition, we demonstrate that phosphomevalonate kinase is a peroxisomal protein which requires the C-terminal peroxisomal targeting signal, Ser-Arg-Leu, for localization to the organelle. -

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. The peroxisomal targeting signals for phosphomevalonate kinase and isopentenyl diphosphate isomerase have been identified. In the current study we identify the peroxisomal targeting signals required for four other enzymes of the cholesterol biosynthetic pathway: acetoacetyl-CoA (AA-CoA) thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, mevalonate diphosphate decarboxylase (MPPD), and farnesyl diphosphate (FPP) synthase. Data are presented that demonstrate that mitochondrial AA-CoA thiolase contains both a mitochondrial targeting signal at the amino terminus and a peroxisomal targeting signal (PTS-1) at the carboxy terminus. We also analyze a new variation of PTS-2 sequences required to target HMG-CoA synthase and MPPD to peroxisomes. In addition, we show that FPP synthase import into peroxisomes is dependent on the PTS-2 receptor and identify at the amino terminus of the protein a 20amino acid region that is required for the peroxisomal localization of the enzyme. These data provide further support for the conclusion that peroxisomes play a critical role in cholesterol biosynthesis.