McAlister-Henn L, Steffan J S, Minard K I, Anderson S L
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284, USA.
J Biol Chem. 1995 Sep 8;270(36):21220-5. doi: 10.1074/jbc.270.36.21220.
The malate dehydrogenase isozyme MDH3 of Saccharomyces cerevisiae was found to be localized to peroxisomes by cellular fractionation and density gradient centrifugation. However, unlike other yeast peroxisomal enzymes that function in the glyoxylate pathway, MDH3 was found to be refractory to catabolite inactivation, i.e. to rapid inactivation and degradation following glucose addition. To examine the structural requirements for organellar localization, the Ser-Lys-Leu carboxyl-terminal tripeptide, a common motif for localization of peroxisomal proteins, was removed by mutagenesis of the MDH3 gene. This resulted in cytosolic localization of MDH3 in yeast transformants. To examine structural requirements for catabolite inactivation, a 12-residue amino-terminal extension from the yeast cytosolic MDH2 isozyme was added to the amino termini of the peroxisomal and mislocalized "cytosolic" forms of MDH3. This extension was previously shown to be essential for catabolite inactivation of MDH2 but failed to confer this property to MDH3. The mislocalized cytosolic forms of MDH3 were found to be catalytically active and competent for metabolic functions normally provided by MDH2.
通过细胞分级分离和密度梯度离心发现，酿酒酵母的苹果酸脱氢酶同工酶MDH3定位于过氧化物酶体。然而，与在乙醛酸途径中起作用的其他酵母过氧化物酶体酶不同，发现MDH3对分解代谢物失活具有抗性，即在添加葡萄糖后快速失活和降解。为了研究细胞器定位的结构要求，通过对MDH3基因进行诱变，去除了过氧化物酶体蛋白定位的常见基序——丝氨酸-赖氨酸-亮氨酸羧基末端三肽。这导致MDH3在酵母转化体中定位于细胞质。为了研究分解代谢物失活的结构要求，将来自酵母细胞质MDH2同工酶的12个残基的氨基末端延伸添加到过氧化物酶体形式和错误定位的“细胞质”形式的MDH3的氨基末端。先前已证明这种延伸对于MDH2的分解代谢物失活至关重要，但未能赋予MDH3这种特性。发现错误定位的细胞质形式的MDH3具有催化活性，并且能够执行通常由MDH2提供的代谢功能。
