Development of the plate assay screening procedure for isolation of the mutants deficient in inactivation of peroxisomal enzymes in the yeast Yarrowia lipolytica

The amine oxidase (AMO) plate assay screening procedure for isolation of the mutants deficient in inactivation of peroxisomal enzymes in the yeast Y. lipolytica has been developed. The first tagged mutants affected in the peroxisomal AMO and isocitrate lyase inactivation were generated by the insertion of a zeta-URA3 mutagenesis cassette into the genome of a zeta-free and игаЗ deletion mutant strain of Y. lipolytica.

Introduction.Extensive peroxisome proliferation du ring growth on oleic acid, combined with the avai lability of excellent genetic tools, makes the dimorphic yeast Y. lipolytica a powerful model system for studying the molecular mechanisms involved in pero xisome biogenesis [1].In glucose medium, pero xisomes become dispensable for growth and undergo autophagic vacuolar-depending degradation [2 ].How ever, the mechanisms by which peroxisomes of Y. lipolytica are taken up by vacuoles during glucoseinduced degradation have not been established [3].Mutants defective in different steps of this process can be used as a powerful approach for elucidation of the mechanisms of pexophagy, although methods for selection of such mutants in Y. lipolytica are not developed in details.Therefore, the aim of this study was the development of the plate assay screening procedure for isolation of the У. lipolytica mutants affected in peroxisomal enzymes inactivation in glu cose medium.
Mutant isolation.The insertional mutagenesis by MTC was performed as previously described [4 ].The mutants deficient in the inactivation of peroxisomal enzymes were isolated by a plate assay screening procedure (Fig. 2), developed on the basis of: 1) qualitative determination of amine oxidase (AMO) in yeast colonies [7]; 2) plate colony assay for visua-Results and Discussion.Development of the plate assay screening procedure.AMO is known to be the strictly regulated peroxisomal enzyme of Y. lipolytica that can be induced by ethylamine and strongly repressed by ammonium ions [2 ].Besides, the expo sure of acetate/oleic acid/ethylamine-grown cells to the glucose excess led to a rapid inactivation of the AMO activity [2].Using the peroxisomal AMO as a reporter enzyme, the plate assay screening procedure for isolation of the mutants deficient in the inacti vation of peroxisomal enzymes was developed (Fig. 2).First, conditions were optimized for peroxisome biogenesis and AMO induction, and for the qualitative plate assay of AMO activity in yeast colonies.The strongest and most reproducible purple coloring was obtained after 18 h incubation of yeast colonies on the

Fig. 1. Restriction map of the JMP5 plasmid with zeta-URA3 mutagenesis cassette
and the MTC specific primers MTC1 and MTC2 <#), which correspond to the right and left borders of MTC fragment excised by Not! from JMP5 lization of the alcohol oxidase activity in H. poly morphs [8 ].For mutant isolation, Ura* transformants from YNBD plates were replica plated onto YYEE plates.After incubation for 18 h, the plates were carefully overlaid with 7-8 ml of 0.3 % top agar AMO inactivation mixture with glucose (3 %) and ammonium sulfate (1 %).After 10 h of incubation at 28 °С the plates were overlaid with 7-8 ml of 0.3 % top agar AMO assay mixture containing 100 mM phosphate buffer (pH 7.0), o-dianisidine (0.05 %) as a chromogen, cetyltrimethylammonium bromide (СТАВ) (0.5 %) to permeabilize cells, peroxidase (2.3 u/ml), and 4 mM ethylamine as an AMO substrate.Reddish colored colonies due to the pre sence of high residual AMO activity were identified after 14 h of plate incubation at 28 °С.
Biochemical methods.Cell-free extracts for en zyme assays were prepared as in [9].Protein con centrations were determined according to [10], using bovine serum albumin as the standard.AMO (EC 1.4.3.4) was assayed as in [11], using the pH 7.0 phosphate buffer and 10 mM ethylamine as the AMO substrate.Isocitrate lyase (ICL) (EC 4.1.3.1) was assayed according to [12].Next, the procedure for inactivation of peroxi somal AMO in yeast colonies was elaborated.All approaches developed previously gave poor results with Y. lipolytica colonies.
For example, inactivation by replica plating on glucose medium, used by Titorenko et al. [8] could not be applied in case of dimorphic colonies of У. lipolytica, because of insufficient amount of biomass for determination of the AMO activity even after 6 h incubation of replicated colonies on the fresh YYEE medium.The inactivation by overlaying induction plates with liquid glucose medium and its subsequent removing after 6-8 h, developed by Gunkel et al. [2], is not acceptable in case of YYEE, the alcohol medium without oleic acid, because of washing out the colony material.To solve this problem, we developed an approach of double overlaying (Fig. 2).The YYEE plates should be overlaid, first, with 0.3 % top agar AMO inactivation mixture with 3 % glucose (to induce pexophagy) and 1 % ammonium sulfate (to repress de novo AMO synthesis).It was previously shown that ammonia ions of such concentration had no inhibitory effect on AMO activity when added to the activity assay mixture for H222-S4 cells induced on the YYEE medium.After appropriate incubation time, the plates should be overlaid with 0.3 % top agar AMO assay mixture with doubled concentrations of phosphate buffer and ethylamine (as in «Materials and Methods»).
Using this approach, the reliable difference in staining between colonies which were or were not subjected to the AMO inactivation was achieved (Fig. 2).At least 8-10 h incubation at 28 °С between overlaying with the AMO inactivation mixture and subsequent pouring with the AMO assay mixture should be applied for almost total AMO inactivation in the plate assay developed.
Isolation of the mutants deficient in inactivation of peroxisomal enzymes.The tagged mutants affected in the AMO inactivation were generated by insertion of the PCR amplified MTC (Fig. 1) into the genome of H222-S4 strain of Y. lipolytica [4].It has been demonstrated, that the MTC insertion occurs ran domly by non-homologous recombination and that mutant phenotypes are due to the genes' disruption with the integration cassette [4].More than 2000 Ura + transformants were analyzed and 10 clones identified as defective in the AMO inactivation (Ain') after 3 sequential rounds of plate screening.For two of them, namely Ain4 and Ain7, the defect of AMO inactivation was confirmed in liquid culture by de termining the enzyme activity in cell-free extracts (Fig. 3, A).The Ain4 mutant was simultaneously affected in inactivation of the second peroxisomal enzyme, ICL (Fig. З, B).Peroxisome degradation in these strains is the subject of further biochemical and morphological analysis.To identify the disrupted gene in the ain4 mutant the regions flanking the integrated MTC will be sequenced using PCR walking method [4].

Inscrtional mutagenesis \ Overlaying the plate with 0.3 % Overlaying the plate with 0.3 % top top agar (control experiment) agar AMO inactivation mixture Incubation for 10 hat 28 °С and overlaying the plates with 0.3 % top agar AMO assay mixture for 14 h Subsequent analysis of AMO and ICL inactivation in liquid culture and electron-microscopical analysis of peroxisome degradation
Fig. 2. Scheme of the plate assay screening procedure for isolation of the mutants deficient in inactivation of peroxisomal enzymes in the yeast Y. lipolytica