Stasyk O. Molecular mechanisms of carbon catabolite regulation and peroxisome homeostasis in methylotrophic yeasts

The main topic of this dissertation work concerns elucidating molecular components involved in regulation of peroxisome biogenesis and autophagic degradation, signaling mechanisms that maintain their homeostasis, sensing of hexose compounds and related catabolite regulation. Yeasts are convenient eukaryotic models for such cell biology research. It is of a significant fundamental and practical interest as some aspects of the obtained knowledge can be translated to human health and various biotechnological processes. 43 Methylotrophic yeasts have a number of advantages for studies on catabolite regulation. Like most other so-called "non-conventional yeasts", they are obligatory aerobes whose signaling mechanisms are not adapted to fermentative growth. Synthesis of peroxisomal and cytosolic enzymes of methanol utilization as well as peroxisome prollifgeration are induced by methanol but is strictly repressed by sugars and ethanol, - effector molecules that rely on distinct catabolic pathways. Catabolite inactivation of peroxisomal enzymes by glucose or ethanol involves degradation of organelles in vacuoles via pexophagy. Signaling and structural mechanisms providing selectivity for autophagy, pexophagy in particular, still remaine not fully elucidated. We demonstrated that glucose signaling in the mechanism of transcriptional induction of glucose transporters in H. polymropha is mediated by a non-transporting sensor Hxs1 (Hexose sensor), whereas the signaling for transcriptional repression in H. pylymorpha is rather "non-conventional" and depends on glucose transport and on the unique to this species protein Gcr1 (Glucose catabolite repression), - a potential transceptor (transporting receptor), which also possesses a regulatory function in the absence of glucose. It has also been established that putative homologs of transcriptional factors of the main repression pathway in S. cerevisiae: Mig1, Mig2 and Tup1, are not the essential components of glucose repression mechanism in H. pоlymorpha. It was also observed that Gcr1 and Hxs1 do not directly participate in the signaling for pexophagy. However, the MIG1, MIG2 and TUP1 gene products are necessary for the physiological regulation of the autophagy type in response to exogenous stimuli. Based on the H. polymorpha mutants with aberrant glucose regulation and developed new approaches for multicopy vector integration, producers of a number of recombinant proteins of medical and biotechnological significance have been constructed. The proposed modified expression platform relies only on sugar substartes for regulation of recombinant protein production. Another goal was to identify new genetic elements controlling pexophagy, paying main atention on those not involved in general autophagy. A collection of P. pastoris pexophagy-deficient mutants has been isolated and a positive selection method for the cloning of the affected genes by functional complemetation elaborated. By functional analysis of mutants in several newly identified pexophagy genes in P. pastoris and H. polymorpha it has been established that function of the product of the ATG26 gene – ergosterolglucosyl transferase, is selectively required only for pexophagy, but not for the general autophagy, and is conserved in these two methylotrophs. It was also found that the product of the P. pastoris ATG28 gene is one of the components of the autophagic apparatus responsible for selective recognition of peroxisomes. A novel peroxisome membrane protein Pex36 has been identified as necessary for both peroxisome biogenesis and degradation in P. pastoris and H. polymorpha. Our data highlight the importance of comparative studies on signaling mechanisms in different yeast species from the point of view of both, fundamental science and biotechnological applications.