Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica

Abstract

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3'-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.

Figure 1.

(A) Phylogenetic tree of the MDHs from 11 fully sequenced hemiascomycetous yeast species using the unique MDH gene from S. pombe (SPCC306.08) as an outgroup. The yeasts are Candida glabrata (CAGL), D. hansenii (DEHA), Kluyveromyces lactis (KLLA), Lachancea thermotolerans (KLTH), Lachancea kluyveri (SAKL), Zygosaccharomyces rouxii (ZYRO), Eremothecium gossypii (ERGO), P. pastoris (Pipas), A. adeninivorans (ARAD), Y. lipolytica (YALI) and S. cerevisiae. The blue zone indicates genes with a typical mitochondrial targeting sequence and the pink zone indicates genes with a potential PTS. The percentages of amino acid identity and similarity among the proteins of each group (peroxisomal, mitochondrial and cytosolic) are indicated next to each group. (B) In silico predictions of protein targeting. The MITOPROT score, PTS1 predictor score and C-terminal sequence are indicated for each MDH.

Figure 2.

Gene model for YlMDH2. (A) Schematic representation of alternative transcripts from the multi-intronic MDH gene YlMDH2. Exons are represented by grey rectangles and introns are symbolized by thin black articulated lines. Vertical bars on each of the three phases (0, +1 and +2) represent in-frame stop codons. (B) Sequence representation of the 3′ regions centred on the second intron. Coloured circles indicate the 5′-splice site, the BP and the two 3′-splice sites used to generate the two mRNA variants. Exon parts are represented by grey rectangles and the two putative C-terminal protein sequences are indicated.

Figure 3.

Expression of alternatively spliced forms of YlMDH2 and growth on different substrates. Serial dilutions (serial dilution factor of five) of cultures of the wild-type (wt—JMY2428) strain, the cytosolic variant (cyto—JMY2416) and the peroxisomal variant (pero—JMY2426) were inoculated on YNB medium supplemented with different carbon sources. No growth differences between the mutants were detected; both of them were able to grow on all the media.

Figure 4.

Comparative growth of YlMDH2 mutants. (A) Growth curves in flasks with agitation in YNBE medium with glucose 2%. (B) Growth curves on 96-well plates in YNBE with glucose 0.5%. Coloured curves represent the different splicing mutants of YlMDH2: the cytosolic form is in black (MDHc), the peroxisomal form is in red (MDHp) and the wild-type is in green (MDHwt). OD, optical density measured at 600 nm.

Figure 5.

Colocalization of the two YlMdh2p isoforms with cytosolic or peroxisomal forms of the RedStar2 protein. eYFP-tagged peroxisomal YlMdh2p (eYFP-MDHp), cytosolic YlMdh2p (eYFP-MDHc) or wild-type YlMdh2p (eYFP-MDHwt) were co-expressed with either the peroxisomal (redstar2p) or cytosolic (redstar2c) forms of the RedStar2 protein. For each strain, both proteins (MDH and RedStar2) were visualized simultaneously by fluorescence microscopy. Cells were imaged after 12h of growth in YNBE 2% OA using differential interference contrast (DIC) for eYFP fluorescence (eYFP, green) and for RedStar2 fluorescence (redstar2, red). eYFP and RedStar2 images were merged (right panel). Yellow colour indicates overlapping fluorescence and evidences colocalization.