Abstract
cDNA encoding for carnitine acetyltransferase (CAT) of yeast S. cerevisiae was isolated by screening a yeast cDNA lambda gt11 library with antibody. The whole coding sequence was obtained from the cDNA and from a YEP 13 DNA clone identified using the cDNA as probe. The coding sequence consists of 670 residues, which amounts to a molecular mass of 77,300 kDa. This cDNA was used successfully to disrupt the gene for the mitochondrial isoenzyme of CAT, which was shown by measuring the enzyme activity and by immunoblot. The acetylcarnitine content of these cells decreased significantly. A search in the PIR protein data base revealed that besides the known carnitine acyltransferases, choline acyltransferases are highly homologous to yeast CAT. The mitochondrial CAT-deficient (CAT-) cells were able to grow on different fermentable and nonfermentable carbon sources, even on acetate at the same rate as the parental strain. In contrast to these, 13C NMR studies revealed significant differences between parental and CAT- cells. In CAT-cells [3-13C]pyruvate was converted mainly to lactate and acetate, whereas in the parental cells alanine and tricarboxylic acid cycle intermediates were found as the main products of pyruvate metabolism beside acetate. These results suggest diminished flux through the pyruvate dehydrogenase complex in the absence of mitochondrial CAT in yeast cells.
Highlights
From the Slnstitute of Biochemistry, University Medical Schoolof Pecs, Szigeti 12, H-7624Pecs, Hungary, the lllnstitut fur Physiologische Chemie, Uniuersitat Munchen, Goethestr3. 3, 0-8000 Miinchen2, Germany, and the )IDepartment of Zoology, Janus Pannonius University, Pees Ifjusag 6, Hungary cDNA encoding focrarnitinaecetyltransferase acetyltransferase (CAT)’is specific for short chain fattaycids (CAT) of yeast S. cerevisiae was isolated by screening andhasthe highest activitywith acetylmoiety (4).CAT a yeast cDNA Xgtl 1library with antibody
The function of CAT has not been described in as great detail as has thatof carnitine palmitoyltransferase or carnitineoctanoyltransferase.Inmammalian cells CATissupposed to buffer againstrapidchanges in acetyl-coA, preventing the depletionof free CoA; on the other hand the acyltransferases, choline acyltransferases are highly product of this enzyme, the acetylcarnitine, can serve as an homologous toyeast CAT
CAT may participate in the deficient ( C A T ) cells were able to grow on different elimination of xenobiotic fatty acids as well, since CAT can fermentable and nonfermentable carbon sources, eventransfer short, branched chain fatty acid to carnitine, on acetate at the same rateas the parental strain
Summary
Gyula Kispal$$, BalazsSumegiS, Klaus Dietmeiery,Ildiko Bock$, Gabriella Gajdosl), Tihamer TomcsanyiII,and Attila Sandor$. CAT may participate in the deficient ( C A T ) cells were able to grow on different elimination of xenobiotic fatty acids as well, since CAT can fermentable and nonfermentable carbon sources, eventransfer short, branched chain fatty acid to carnitine, on acetate at the same rateas the parental strain. Tricarboxylic acid cycle intermediates were found as In lower eukaryotes, the transport of the acetyl moiety in the main products of pyruvate metabolism beside ace- or out of the mitochondria has beenconsidered as a possible tate These results suggest diminished flux through thfeunction for CAT.Inthealkane growing yeast, Candida pyruvatedehydrogenase complex intheabsence of tropicalis acetyl-coA forms in thceytosol, so acetyl group can mitochondrial CAT in yeast cells. This article were defrayed in part by the payment of page charges
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