AZF1P IS A NUCLEAR-LOCALIZED ZINC-FINGER PROTEIN THAT IS PREFERENTIALLY EXPRESSED UNDER NON-FERMENTATIVE GROWTH-CONDITIONS IN SACCHAROMYCES-CEREVISIAE

In previous studies the AZF1 gene has been identified as a second high -copy number suppressor for a special mutant of the gene for the mitoc hondrial core enzyme of RNA polymerase. The first high-copy number sup pressor of this mutant turned out to be the specificity factor MTF1 fo r mitochondrial transcription. Up to now, the influence of AZF1 on mit ochondrial transcription, its precise localization in the cell and the regulation of its expression has not been determined. The putative pr otein contains a long stretch of poly-asparagine amino acids and a typ ical zinc-finger domain for DNA binding. These characteristic structur al features were used to create the abbreviation AZF1 (Asparagine-rich Zinc Finger protein). An initial computer analysis of the sequence ga ve no conclusive results for the presence of a mitochondrial import se quence or a typical nuclear-targeting sequence. A recent more-detailed analysis identified a possible nuclear localization signal in the mid dle of the protein. Disruption of the gene shows no effect on plates w ith glucose-rich medium or glycerol. In this report a specific polyclo nal antibody against Azf1p was prepared and used in cell-fractionation experiments and in electron microscopic studies. Both of these clearl y demonstrate that the AZF1 protein is localized exclusively in the nu cleus of the yeast cell. Northern analysis for the expression of the A ZF1 messenger RNA under different growth conditions was therefore perf ormed to obtain new insights into the regulation of this gene. Togethe r with the respective protein-expression analysis these data demonstra te that Azf1p is preferentially synthezised in higher amounts under no n-fermentable growth conditions. Over-expression of Azf1p in the yeast cell does not influence the expression level of the mitochondrial tra nscription factor Mtf1p, indicating that the influence of Azf1p on the suppression of the special mitochondrial RNA polymerase mutant is an indirect one. Subcellular investigation of the deletion mutant by elec tron microscopy identifies specific ultrastructural cell-division defe cts in comparison to the wild-type.