Tatsuyuki Kamiryo

New aspects of sterol carrier protein 2 (nonspecific lipid-transfer protein) in fusion proteins and in peroxisomes

Sterol carrier protein 2 (SCP2) is a 13-kDa peroxisomal protein, identical to nonspecific lipidtransfer protein, and stimulates various steps of cholesterol metabolism in vitro. Although the name is reminiscent of acyl carrier protein, which is involved in fatty acid synthesis, SCP2 does not bind to lipids specifically or stoichiometrically. This protein is expressed either as a small precursor or as a large fusion (termed SCPx) that carries at its C-terminal the complete sequence of SCP2. SCPx exhibits 3-oxoacyl-CoA thiolase activity, as well as sterol-carrier and lipid-transfer activities. The N- and C-terminal parts of SCPx are similar to the nematode protein P-44 and the yeast protein PXP-18, respectively. P-44, which has no SCP2 sequence, thiolytically cleaved the side chain of bile acid intermediate at a rate comparable to that of SCPx. This, together with the properties of other fusions with SCP2-like sequence, suggests that the SCP2 part of SCPx does not play a direct role in thiolase reaction. PXP-18, located predominantly inside peroxisomes, is similar to SCP2 in primary structure and lipid-transfer activity, and protects peroxisomal acyl-CoA oxidase from thermal denaturation. PXP-18 dimerized at a high temperature, formed an equimolar complex with the oxidase subunit, and released the active enzyme from the complex when the temperature went down. This article attempts to gain insight into the role of SPC2, and to present a model in which PXP-18, a member of the SCP2 family, functions as a molecular chaperone in peroxisomes.

Metabolic significance and expression of Caenorhabditis elegans type II 3-oxoacyl-CoA thiolase

The authors cloned the cDNA of the nematode Caenorhabditis elegans encoding a 44-kDa protein (P-44), which is similar to sterol carrier protein x (SCPx). Genomic DNA data and Northern blot analysis excluded the possibility of P-44 forming SCPx-like fusion protein. P-44 is required in the formation of bile acid in vitro from CoA esters of their enoyl-form intermediate in the presence of d-3-hydroxyacyl-CoA dehydratase/d-3-dehydrogenase bifunctional protein. Also, rat SCPx converts 24-hydroxy-form intermediate to bile acid under similar conditions. From this and other evidence, P-44 and SCPx were categorized as type II thiolase. The mRNA encoding P-44 was detected in every developmental stage of C. elegans: egg, larval stages, and adult. P-44, therefore, seems essential for the normal functioning of this organism.

Isolation of Saccharomyces cerevisiae mutants defective in acyl coenzyme a synthetase.

Mutants of Saccharomyces cerevisiae defective in acyl-CoA synthetase (EC 6.2.1.3) were isolated. The mutants were concentrated by the radiation-suicide technique with the use of tritiated palmitic acid. Selection of the mutants was based on the premise that acyl-CoA synthetase activity would become indispensable when yeast cells in which fatty acid synthesis de novo is blocked are grown in a medium supplemented with fatty acid. The mutant strains isolated exhibited low acyl-CoA synthetase activity in vitro. Furthermore, they accumulated markedly more of the incorporated palmitic acid in the nonesterified form than did the wild- type strain. Some of the mutants showed thermosensitive acyl-CoA synthetase activity, indicating a mutation of the structural gene of the enzyme. Genetic studies on these mutants indicated that their phenotype resulted from a single, recessive mutation of a nuclear gene, designated faa 1 (fatty acid activation).