SenSing and Signaling for PeroxiSome autoPhagic degradation ( PexoPhagy ) in yeaStS

yeast cells, similarly to cells of other eukaryotic organisms, possess intracellular organelles, including that of peroxisomes also known as microbodies . Enzymes of oxidative metabolism, mainly hydrogen peroxide generating oxidases, catalase, some enzymes of glyoxylic cycle and enzymes involved in catabolism of unusual carbon sources (n-alkanes, methanol) are located in peroxisomes. Especially important role is played by peroxisomes in methylotrophic yeasts, unique eukaryotic organisms capable to utilize one-carbon compound, methanol. Active proliferation and biogenesis of peroxisomes occur on methanol, so these organelles can occupy between 30 and 80% of cellular volume. After shift of methanol-grown cells into media with multicarbon substrates, such as glucose or ethanol, an excess of peroxisomes degrades in the specific process known as autophagic degradation of peroxisomes or pexophagy. There are 36 AuTophaGy related genes, known as ATG genes, which products are also involved in pexophagy. At the same time, not much is known on mechanisms of glucose and ethanol sensing and signaling which initia te pexophagy process. Proteins Pfk1(α-subunit of phosphofructokinase), Slt2 (mitogen-activating protein kinase) Gpr1 and Gpa2 (components of GPCr system) and Snf3 and Ggt2 (highand low-affinity glucose sensors) were found to be involved in signaling of glucose-induced pexophagy in Saccharomyces cerevisiae. In the methylotrophic yeast Pichia pastoris, glucose sensing protein Gss1 was found to be important for glucose-induced pexophagy. Very few is known on mechanisms of ethanol sensing and signaling during pexophagy which is an important problem for future studies.


M
ethylotrophic yeasts are unique euka ryotic organisms capable of utilizing onecarbon toxic substrate, methanol.During methylotrophic growth, peroxisomes oc cupy 3080% of cellular volume.Shift of methy lotrophicallygrown cells to media with alterna tive carbon sources, glucose or ethanol, induces massive peroxisome degradation.In Pichia pastoris, two morphologically distinct events have been observed, macro and microautophagy; in other species, mostly macroautophagy was noted under massive peroxisome degradation.It was found that genes involved in nonspecific autophagy (most of them are known as ATG genes) also participate in carboninduced pexophagy.Many ATG genes have been discovered on the models of methylotrophic yeasts, mainly P. pastoris, due to convenient and easy methods for pexophagy monitoring.However, mechanisms of glucose and ethanol sensing and signaling which initiate subsequent events of mic ro and macroautophagy are poorly understood.Also the nature of the lowmolecularweight ef fectors, derivatives of glucose and ethanol, which induce pexophagy, has not been identified.
It was found that P. pastoris possesses a single glucose sensor Gss1, ortholog of S. cerevisiae high and lowaffinity glucose sensors Snf3 and Rgt2, respectively.Gss1 protein participates in glucose sensing involved in pexophagy and glucose catabo lite repression.In contrast to Saccharomyces cerevisiae, P. pastoris orthologs of GPCR signaling pro teins Gpr1 and Gpa2 do not participate in glucose signaling of pexophagy.It is known that one of the signal proteins participating in micropexophagy in P. pastoris is αsubunit of phosphofructokinase Pfk1, whose catalytic activity is not necessary for glucose induced micropexophagy.The role of Slt2, mitogenactivated protein kinase (MAPK), was also revealed in glucose signaling of pexophagy.Ethanol signaling was studied in mutants defective in ethanol catabolism of the yeast P. methanolica.It was suggested that in the medium with ethanol, glyoxylic acid is the substance which triggers pex ophagy.

Peroxisomes and their functions
Peroxisomes are ubiquitous organelles present in virtually all eukaryo tic cells, with exception of Archaezoa (Michels et al., 2005;Brown and Baker , 2008).Peroxisomes also known as microbodies (specific types of these organelles are also named as glyoxysomes and glycosomes) are organelles BIoCHEMISTRy AnD BIoTECHnoloGy foR MoDERn MEDICInE surrounded by a single membrane, their size is of 0.5-1.5 μm at an average.They do not contain DnA, RnA and ribosomes.Cell can contain from 12 peroxisomes (e.g.yeast growing on glucose) to seve ral hundred peroxisomes as mammalian cells (Till et al., 2012).According to the name, per oxisomes harbor H 2 o 2 producing oxidases and decomposing latter compound catalase.However , peroxisomes are extremely versatile organelles sometimes specializing in different functions.An interesting peculiarity of peroxisomal catabolizing enzymes is their inability to produce ATP, which distinquishes them from catabolic enzymes located in mitochondria (Mast et al., 2010).liver peroxi somes contain enzymes that enable to metabolize both verylongchain fatty acids and βoxidation of fatty acids and bile acid precursors with the oxida tion of ingested ethanol to acetaldehyde to account for as much as 50% of the total metabolism of eth anol when substrates for the branchedchain fatty acids , phytanic acid and lipidbased xenobiotics.In yeasts, peroxisomes are responsible for initial steps of methanol and fatty acid catabolism (Veenhuis et al., 1983; van der Klei et al., 2006).In addition to catabolic, peroxisomes fulfill biosynthetic func tions.In mammals, peroxisomes harbor enzymes participating in synthesis of bile acids, cholesterol and plasma logens (Wanders et al., 2010).In myce lial fungi, peroxisomes are involved in lysine bio synthesis in yeasts and penicillin biosynthesis in mycelial fungi (Schrader and fahimi, 2008;Aksam et al., 2009;Meijer et al., 2010).In parasitic pro tozoa of the genera Trypanosoma and Leishmania, glycolytic enzymes occur in a specialized peroxi some, which is known as glycosome (Michels et al., 2006).The compartmentalization of glycolytic enzymes is essential for the survival of these pro tozoa.Voronin bodies, which serve to plug septal pores in mycelial fungi, are also specialized perox isomes.Plant peroxisomes are classified into three groups: glyoxisomes, leaf peroxisomes, and unspe cialized peroxisomes.There are approximately 50 proteins in animal and fungal peroxisomes and approximately 100 proteins in plant peroxisomes.Proteomic and genetic studies continuously re veal new functions for peroxisomes (Michels et al. 2005;lanyonHogg et al., 2010).
Defects in peroxisome structure and functions underlie many human diseases.The so called Zell weger syndrome is the best known peroxisomal in heritable disease.Patients with Zellweger syndrome fall into four groups with different defects in pro tein transport to peroxisomes.The defects occur in peroxisomal protein transport, which involves only peroxisome targeting signal 1 (PTS1), only PTS2, both PTS1 and PTS2, or the two (PTS1 and PTS2) protein translocation pathways and per oxisomal membrane biogenesis (Subramani, 1997).Peroxisome damage has serious consequences and is often fatal, causing death within the first year of life (Steinberg et al., 2006).It is of interest that identical genetic defects were observed in yeasts with distorted peroxisome biogenesis (so called pex mutants) (Subramani, 1998).In summary, peroxi somes are surprisingly dynamic organelles, whose dimensions, number in the cell, and protein con tent change in response to environmental changes.Peroxisome biogenesis is accompanied by other processes, including signal transduction (Saleem et al., 2008), chromatin modification (Wan et al., 2011), reorganization of transcription networks (Smith et al., 2002), and changes in the peroxiso mal proteome (Marelli et al., 2004;Saleem et al., 2006).
yeasts provide a convenient model to study the mechanisms of peroxisome biogenesis because cell transfer from a glucosecontaining medium into a medium with oleate and/or methanol in the case of methylotrophic yeasts induces synthe sis of peroxisomal enzymes and the growth and division of peroxisomes.Peroxisomes may occupy up to 80% of the cell volu me in cells growing in the presence of methanol under certain conditions (Veennuis et al., 2003;Sibirny, 2012).When cells growing in the presence of methanol or oleate are transferred into a glucosecontaining medium or from methanol to ethanolcontaining medium, the transfer is rapidly followed by autophagic deg radation of the majority of peroxisomes (pexo phagy), while one peroxisome somehow avoids this degradation in a way that is still unclear (Dunn et al., 2005).Methylotrophic yeasts appear to be one of the most convenient models for studying peroxisome biogenesis and degradation due to abil ity of methanol to induce massive propagation of peroxisomes.As a result, one or two small peroxi somes present in cells during growth in glucose are substituted by numerous large peroxisomes which occupy near 30% of cell volume during batch culti vation and up to 80% of cell volume under contin uous cultivation under low dilution rate in metha nol as sole carbon and energy source (Veenhuis et al., 2003).Inverse shift of methanolgrown cells to glucose (or ethanol) causes major reorganization of intracellular structure leading to degradation of the majority of peroxisomes due to autophagic pro cess; consequently, from 30 to 80% of cell volume is degraded.Methods of classical and molecular genetics are well developed for several species of methylotrophic yeasts (Cregg et al., 2008;faber et al., 1995;lahtchev et al., 2002;Tolstorukov et al., 2007) and genome sequence of several type strains are publicly available (http://www.genome.jp/keggbin/show_organism?org=ppa or http:// www.pichiagenome.org/ for P. pastoris and http:// genomeportal.jgipsf.org/Hanpo2/Hanpo2.info.html for H. polymorpha).Thus, available tools per mit mecha nistic description of events which occur during autophagic degradation of peroxisomes in methylotrophic yeasts.
Autophagy of cytosolic cell components mostly occurs due to nonspecific process though specific autophagy is proved to be responsible for degradation of fructose1,6bisphosphatase and malate dehydrogenase in the baker's yeast S. cerevisiae.The shift of methylotrophic yeasts from methanol to glucose medium leads, in addition to autophagy degradation of peroxisome (pexophagy), to inactivation of cytosolic enzymes of methanol metabolism (formaldehyde dehydrogenase, formate dehydrogenase, fructose1,6bisphosphatase) and fAD synthesis (riboflavin kinase, fAD synthetase) (Brooke et al., 1986).Inactivation of fructose1,6 bisphosphatase in P. pastoris apparently occurs due to degradation process (o.Dmytruk, A. Sibirny, unpublished).However, it is not known till now where the mentioned enzyme inactivation is a re sult of autophagic process.
Pexophagy can occur as part of nonspecific general autophagy mechanism.Apparently it takes place during yeast propagation in each medium as a component of cell constituent maintenance, housekeeping or turnover mechanism (Aksam et al., 2007).However, massive pexophagy occurs during the shift from some cultivation conditions to other ones.The last type of pexophagy is the specific one.Peroxisome degradation in H. polymorpha, similarly to mammal cells, could also oc cur in the process which is unrelated to autophagy, but involves permeabilization of the peroxisomal membrane mediated by 15lipoxygenase (Baerends et al., 1996;yokota, 2003).Upon lysis, the con tents of the peroxisome become digested by cy tosolic proteases.In H. polymorpha, such kind of peroxisome disintegration was observed in a con structed strain where the levels of the peroxin Pex3 had been strongly reduced.This suggests that loss of certain peroxisomal membrane proteins may destabilize the peroxisomal membrane, resulting in its lysis.Genes involved in pexophagy in methy lotrophic yeasts are homologous to those found in S. cerevisiae (van Zutphen et al., 2008;Polupanov et al., 2011;Till et al., 2012;Suzuki, 2013).
The methods for isolation of the mutants defective in pexophagy have been developed in methylotrophic yeasts.All of them belong to nega tive selection methods when a few mutant colonies grow on plates among a huge number of wild type colonies, and mutants are identified afterwards di rectly in colonies using peroxisome enzyme analy sis (Stasyk et al., 2008a).Apart from mutagene sis under standard mutagen treatment, the insertion mutagenesis using DnA fragments was proposed, which substantially facilitates further cloning of mutant genes (Mukaiyama et al., 2002).
Most of steps and genes involved in spe cific pexophagy also participate in general (non specific ) autophagy.The steps of autophagy and participating genes are as follows (Manjithaya et al., 2010) (fig.1).
8. Transport and heterotypical fusion of au tophagosome and vacuoles (v and tSnAREs, Ccz1, Mon1, and HoPS complex).P. pastoris ATG28 also encodes pexophagy specific protein as its deficiency impairs both pexophagic mechanisms (macro and micropexo phagy) and only partially affects the general (non specific bulk turnover) autophagy induced by ni trogen starvation (Stasyk et al. 2006b;nazarko et al., 2009).Atg28 contains a coiledcoil domain that overlaps with a putative leucine zipper motif.This coiledcoil region in Atg28 may be involved in oligomerization and proteinprotein interac tions.It is functionally important, as modified Atg28 lacking coiledcoil is not functionally ac tive.Atg28 is involved in the formation of one or more protein complexes specific for pexophagy and its interaction with micropexophagyspecific protein Atg35 was experimentally proved (nazarko et al. 2011, see below).Atg28 exhibits a complex intracellular localization pattern.In most metha nolinduced cells, this protein was localized to the cytosol.However, in some cells, the fusion protein was also localized to punctate structures of un known nature associated with vacuoles and to the vacuolar membrane.In rare cases, Atg28 could be seen localized to the vacuolar matrix.
Another pexophagyspecific protein is Atg30.Two other proteins specifically involved in pexo phagy and not in general autophagy or other types of specific autophagy are Pex3 and Pex14, known as peroxins also involved in peroxisome biogenesis.In P. pastoris, Atg30 interacts with two proteins, Pex3 and Pex14, localized on the peroxisomal membrane (farre et al., 2008).Effective peroxi some homeostasis probably requires their biogene sis and degradation to be coordinated.It was shown that interacting partners of Atg30 are pro teins participating in peroxisome biogenesis.Thus, Pex3 is important for peroxisome biogenesis, and Pex14 -for protein import to peroxisomal ma trix (Ma and Subramani 2009).In H. polymorpha Pex14, more exactly the 64 nterminal amino acid residues, are necessary for pexophagy (Bellu et al., 2001a;van Zutphen et al., 2008).Also, it was shown that during macropexophagy in H. polymorpha Pex3 is removed from peroxisomes and does not undergo degradation (Bellu et al., 2002).The way Pex3 is removed from peroxisomes is un known.Pex3 is known to be required for stabiliza tion of a complex of proteins with a RInG finger domain (Really Interesting new Gene, structural domain similar to protein zinc finger) of peroxi some importer (Hazra et al., 2002).Therefore, at this stage, besides inhibi ting peroxisome biogene sis, also destabilization of some complexes in per oxisomal membrane occurs.
In P. pastoris, a gene designated PDG1 (Per oxisome DeGradation) was identified whose muta tions led to disturbances in peroxisome degrada tion (Dunn et al., 2005; o.Stasyk and A. Sibirny, unpublished data).Moreover, such mutations dis turbed localization of peroxisomal proteins that, apart from peroxisomes, were also localized in cytosol, indicating disturbance in peroxisome bio genesis in pdg1 mutants.Corresponding protein Pdg1 is a membrane peroxin, which confirms its role in peroxisome biogenesis.
In H. polymorpha, the transcriptional repres sor Tup1 was shown to be essential for macropex ophagy (leaoHelder et al., 2004).Defects in orthologs of presumable corepressors involved in glucose catabolite repression, MIG1 and MIG2, also showed impairment in pexophagy (Stasyk et al., 2007).As mutants defective in MIG1 and MIG2 were not affected in glucose catabolite re pression, one may assume that functions of these genes are different between baker's and methylo trophic yeasts.

micro-and macropexophagy in Pichia pastoris and Hansenula polymorpha
As was pointed above, macropexophagy could be observed in P. pastoris after shift of methanol grown cells to the medium with ethanol whereas micropexophagy is observed when methanol grown cells are transferred into medium with glucose (Tuttle and Dunn, 1995).other methylo trophic yeast, H. polymorpha, is characterized by macropexophagy independently on carbon source which induces pexophagy (van Zutphen et al., 2008).During macropexophagy, multiple mem brane layers sequester a single peroxisome resulting in the formation of a pexophagosome of which the outer membrane layer fuses with the vacuole where the peroxisome becomes hydrolyzed.Micropex ophagy involves the uptake of a cluster of peroxi somes through direct engulfment by the vacuolar membrane (fig.1).Three main steps could be outlined for macropexophagy: recognition of the organelle destined for degradation, formation of the pexophagosome, and fusion with the vacuole (fig.1).for micropexophagy, the following steps could be distinguished: vacuolar engulfment of peroxisomes, formation of the MIPA at the per oxisomal surface, and vacuolar membrane fusion (Sakai et al., 2006).
Micropexophagy turned out to be more sensi tive to a decrease of intracellular ATP compared to macropexophagy; in other words, intracellular ATP pool plays a more important role in defining BIoCHEMISTRy AnD BIoTECHnoloGy foR MoDERn MEDICInE the pexophagy pathway than the nature of the car bon substrate (Ano et al., 2005b).However, it is not known whether ATP concentration is the rea son of the observed type of pexophagy or is the consequence of some other trigger mechanisms.In other methylotrophic yeasts, e.g.H. polymorpha, shift of methanolgrown cells either to glucose or ethanol leads to morphological changes described as macropexophagy.nitrogen limitation leads in H. polymorpha to peroxisome degradation in by a mechanism similar to micropexophagy.However, this process occurs due to nonspecific autophagic mechanism, as cytosolic components are taken up by vacuoles concomitantly with peroxisomes and therefore was named by authors as microautophagy of peroxisomes (Bellu et al., 2001b;van Zutphen et al., 2008).
During last years, genes specifically involved in macro and micropexophagy have been iden tified.Gene H. polymorpha ATG25 is specifically involved in macropexophagy.It is a coiledcoil protein and acts as the selectivity factor during macropexophagy (Monastyrska et al. 2005).This protein is located in pexophagosomes and moved there via the PAS.Atg25 is involved in the com pletion of sequestration of peroxisomes or in the fusion of pexophagosomes with the vacuolar mem brane (Sakai et al. 2006).for the latter process, the SnARE Vam7 and the GTPase ypt7 are also essential in H. polymorpha (Stevens et al., 2005).
The presence of a specific morphologi cal structure in the micropexophagy process, the micropexophagy apparatus MIPA in P. pastoris, suggests the existence of specific genes and pro teins participating in this process.Gene PFK1 en codes phosphofructokinase 1 αsubunit, which is required for peroxisome engulfment by vacuoles after transferring P. pastoris cells from methanol medium to glucose medium (yuan et al., 1997).Participation of phosphofructokinase 1 αsubunit in micropexophagy does not depend on its abili ty to phosphorylate fructose6phosphate since a catalytically inactive form of this enzyme provides for normal pexophagy.Moreover, the VAC8 gene (VACuole related) was identified whose product is a 6064 kDa protein with so called armadillo repeat that specifically participates in micro but not macropexophagy ( GCN2 encodes protein kinase and regulates trans lation initiation (eIf2 kinase); GCN3 encodes for translation initiation factor (eIf2B), whereas GCN4 encodes for basic leucine zipper (bZIP) transcrip tional activator of amino acid biosynthetic genes in response to amino acid starvation.The exact func tions of the mentioned genes in micropexophagy remain unknown.
The new micropexophagy specific protein Atg35, the first autophagy protein with nuclear localization, was identified during the analysis of partners interacting with protein Atg28 from P. pastoris (Stasyk et al., 2006).To search for such Atg proteins, a yeast two hybrid (yTH) screening system was used for the first time.yTH screening of the genome database of P. pastoris DnA was carried out in S. cerevisiae cells using PpAtg28 as "bait" (nazarko et al., 2011).Two sequences were revealed encoding proteins Atg35 and Rdi1 (Rho GDP Dissociation Inhibitor).Atg35 consists of 463 a.a. and incorporates two putative domains: RInG finger and PHD (Plant Homeo Domain).Testing P. pastoris mutant atg35∆ showed that macropexo phagy is normal whereas micropexo phagy is im paired.Comparison of vacuolar isolating mem brane formation and the micropexo phagy apparatus in wildtype cells and the mutant using fluorescent microscopy revealed that formation of vacuolar isolating membranes in the mutant was normal.The micropexophagy apparatus MIPA was found in atg35∆ cells 1.5 times less frequently than in wildtype cells, while in atg28∆ mutant the micro pexophagy apparatus was not being formed at all.However, both mutants (atg28∆ and atg35∆) exhib ited normal formation of pexophagosomes during macropexophagy, which were not formed at all in atg1∆ mutant.Studying the role of Atg35 in general autophagy and Cvt pathways revealed that this pro tein is not required for either process.Thus, Atg35 is necessary only for micropexophagy at the stage of micropexophagy apparatus formation (nazarko et al., 2011).It is interesting that overexpression of ATG35 as well as deletion of this gene both inhibit micropexophagy but do not influence macropex ophagy.With ATG35 overexpression the forma tion of vacuolar sequestering membranes was not disturbed, while formation of the micropexophagy apparatus was blocked.However, overexpression of ATG35 did not influence general (nonspecific) au tophagy (nazarko et al., 2011).Studying expression of genes ATG28 and ATG35 during peroxisome proliferation and degradation revealed that corre sponding proteins are almost completely absent in ethanol medium though they are present in signifi cant amounts in glucose and methanol medium.
Atg35 contains putative nuclear localization signal.Testing of localization of overexpressed hy brid protein Atg35-eyfP revealed that in metha nol medium it was localized in the nucleus and single dotlike structures on nuclear membrane (perinuclear structure, PnS).Transfer of cells to glucose medium caused homogenous distribution of Atg35-eyfP in the nucleus.However, combined overexpression of eCfP-Atg17 caused relocaliza tion of Atg35-eyfP to single dotlike structures of nuclear membrane during micropexophagy.More over, in glucose medium Atg35-eyfP on dotlike structures of nuclear membrane colocali zed with one of the eCfP-Atg17 dots.obviously eCfP-Atg17 accumulation on nuclear membrane during micropexophagy is necessary for organization of single dotlike structures of nuclear membrane and involvement of Atg35-eyfP into this structure.Supposedly, Atg35 localization on single dotlike structures of nuclear membrane in glucose medium depends on Atg17 and is significant for the micro pexophagy process.
Atg28 is known to interact with Atg17 (nazar ko et al., 2007a) and Atg35 (nazarko et al., 2011).It was showed that interaction between Atg17 and Atg35 occurs due to Atg28 protein (fig.2. figu re 6, from nazarko et al., 2011).Thus, Atg35 is the first revealed nuclear Atg protein participating in autophagy in yeasts.Deletion and overexpression of this gene lead to specific disturbance of micro pexophagy alone.Atg35 protein functions through interaction with Atg17 and Atg28, the latter protein playing a central role in this interaction (nazarko et al., 2011).

glucose sensing and signaling mechanisms and pexophagy in H. polymorpha and P. pastoris
When cells are transferred from methanol to glucose medium, micropexophagy (P.pastoris) or macropexophagy (H.polymorpha) occur.Cells in some way recognize (sense) glucose and triggers glucose signal to activate all specific to micropexo phagy, other pexophagyspecific and many gene ral autophagy Atg proteins ended by peroxisome degradation.Mechanisms of glucose sensing and signaling during (micro)pexophagy is poorly un derstood, especially in methylotrophic yeasts.
Below we consider available data on glucose sensing and signaling connected to pexophagy.
Sensing.Mechanisms of glucose sensing have been studied in detail in S. cerevisiae as glucose induces complex regulatory responses, which in clude induction of glucose transporters, catabolite repression of hundreds of genes, catabolite inacti vation of several enzymes including proteasomal and autophagic degradation of some of them.Be sides, glucose is sensed for subsequent trehalose mobilization and other responses to stress factors.Still many aspects of glucose sensing in S. cerevisiae remain to be elucidated.Reader is referred to corresponding reviews (ozcan and Johnson, 1999; Santangelo, 2006;Gancedo, 2008;RubioTexeira et al., 2010).Briefly speaking, there are two types of glucose sensors in S. cerevisiae.one is involved in glucosedependent stress response and the other one is responsible for glucose induction and glu cose catabolite repression phenomena.
Plasma membrane contains many proteins ca pable of glucose binding and part of them act as glucose sensors.There are 20 glucose transporters (Wieczorke et al., 1999), however, all they appar ently are not involved in glucose sensing (Gancedo 2008).Specific glucose sensors can be divided in three groups.The first class of sensors comprises the classical receptor proteins or G proteincou pled receptors (GPCRs), which, in yeast, detect the presence of glucose and sucrose.It is responsible for glucose and sucrose control of the protein ki nase A (PKA) pathway (Thevelein and de Winde, 1999) which plays a central role in the nutritional control of metabolism, stress resistance, cell cycle, growth, and transcription.All these properties are tightly controlled by the availability of nutrients in the medium, especially by the presen ce of a rap idly fermentable sugar, glucose.Addition of rap idly fermentable sugars to derepressed yeast cells triggers an immediate increase in the cAMP level, which in turn causes rapid activation of PKA, re sulting in drastic changes in its multiple targets.The sugarsensing GPCR system consists of the receptor Gpr1 and the Gα protein Gpa2 (Co lombo et al., 1998).The second class of glucose sensors in S. cerevisiae is represented by two non transporting transceptors Snf3 and Rgt2 which are sugar transporter homologs.Highaffinity sensor Snf3 and lowaffinity glucose sensor Rgt2 generate intracellular signal required for induction of hexose transporter genes in response to glucose (Gancedo, 2008)  In methylotrophic yeasts, we know much less on glucose sensing and signaling.In H. polymorpha, two glucose sensors, Gcr1 and Hxs1, have been identified, along with glucose (hexose) trans porter Hxt1 (Stasyk et al. 2004(Stasyk et al. , 2008b)).P. pastoris, on the other hand, apparently possesses only one glucose sensor, designated as Gss1 (Polupanov et al., 2011).Point or deletion mutations in GCr1 gene of H. polymorpha affected glucose catabolite repression and led to constitutive presence of per oxisomes in glucose medium (Stasyk et al., 2004).However, GCr1 gene apparently is not directly in volved in pexophagy.It was observed a decrease in specific activity and protein levels of peroxiso mal enzyme alcohol oxidase in gcr1 mutant cells upon glucose adaptation, but residual alcohol oxi dase levels were higher in the gcr1 mutants rela tive to the wild type.However, these data do not demon strate a direct involvement of Gcr1 protein in pexophagy since in gcr1 strains, de novo peroxi some synthesis occurred due to the defect in glu cose repression.A time course examination of cell morpholo gy revealed clear signs that pexophagy proceeds in gcr1 mutants.Some peroxisomes were observed sequestered by additional membrane lay ers typical of initial stages of macroautophagic per oxisome degradation in H. polymorpha (Veenhuis et al., 2000).Also, in gcr1 cells with fluorescently labeled peroxisomes, the pexophagic process was evident upon glucose adaptation.Shortly after the shift, GfP fluorescence was observed in vacuoles, while in methanolgrowing cells it is confined to peroxisomes.These data led to the conclusion that Gcr1 is not directly involved in pexophagy.Both point missense and deletion gcr1 mutants contin ued to exhibit normal wildtype peroxisome deg radation in response to ethanol.
Contrary to that, knock out mutation in an other hexose sensor gene HXS1 did not lead to defect in glucose catabolite repression and led to defect in glucose transport capacity (Stasyk et al., 2008b).To study, whether HXS1 is involved in pexophagy, it was observed that in methanol preincubated hxs1Δ cells, alcohol oxidase activity and protein level decreased upon glucose adapta tion with a rate similar to that of the wildtype strain.The H. polymorpha tup1 mutant deficient in pexophagy has been utilized as a positive con trol (leaoHelder et al. 2004;Stasyk et al. 2007).When methanolpreinduced hxs1Δ cells were shift ed to fructose or ethanol, they also did not differ in the rates of alcohol oxidase degradation from the wildtype strain.Therefore, Hxs1, similarly to Gcr1, is not essential for glucose signaling in pex ophagy in H. polymorpha.Thus, both of identified glucose sensors in this organism are involved in several regulatory processes exerted by glucose but not in glucose recognition for pexophagy.So, the specific glucose sensor involved in glucoseinduced macropexophagy in H. polymorpha still needs to be found.
Situation in P. pastoris looks differently.In this organism, orthologs of GPCR sensor proteins Gpr1 and Gpa2 and glucose transceptor sensor proteins Snf3/Rgt2 have been identified.one po tential ortholog of the GPr1 gene and one of the GPA2 gene, that exhibit 60% and 65% similari ty to their S. cerevisiae counterparts, respectively.Com plete GPr1 and GPA2 open reading frames were knocked out by gene replacement method using ScArG4 as a marker gene.Corrected knockouts of corresponding genes were confirmed by PCR.Iso lated P. pastoris Δgpr1 and Δgpa2 mutants as well as strain SMD1163 defective in vacuolar proteases (Tuttle and Dunn, 1995) were used for studying pexophagy.Two kinds of experiments were done.In the first experiment, degradation of peroxisomal thiolase followed after the transfer of oleategrown cells to glucose (2%) medium, i.e., conditions were exactly the same as those which were used for S. cerevisiae.In the second experiment, cells were induced for peroxisome proliferation by incuba tion on methanol (0.5%) medium and then replica plated on the medium with ethanol or glucose.In this case, activity of a key peroxisomal enzyme of methanol metabolism, alcohol oxidase was ana lyzed in situ.It was found that knockout of the putative orthologs of GPr1 and GPA2 in P. pastoris has no apparent effect on both degradation of per oxisomal thiolase and inactivation of peroxisomal alcohol oxidase (fig.4; fig. 3 from nazarko et al., 2008).These mutations also had no effect on gene ral autophagy (nazarko et al., 2008b).Thus, in contrast to S. cerevisiae, PpGpr1 and PpGpa2 are not involved in glucose signaling for pexophagy in P. pastoris.It is known that in contrast to S. cerevisiae, Candida albicans GPr1 and GPA2 are not involved in a transient cAMP burst after glucose addition (Maidan et al., 2005).Similarly PpGpr1 and PpGpa2 could be not involved in regulation of cAMP production and it looks like glucose for pexophagy is sensed in P. pastoris by other com ponents of the PKAcAMP signaling pathway or only by distinct sensors which are not involved in this pathway.
Two hexose transporters were recently iden tified in the yeast P. pastoris, Hxt1 and Hxt2, which are transcriptionally regulated by glucose.Deletion of PpHXT1 but not PpHXT2, led to the expression of alcohol oxidase in glucose medium due to glucose catabolite repression impairment.However, mutant lacking PpHxt1 was normal in both respects, glucose utilization and peroxi some degradation (Zhang et al., 2010).The search for orthologs of S. cerevisiae glucose transceptor sensors SNF3 and rGT2 revealed that P. pastoris contains only one ortholog, designated as GSS1 (from GlucoSe Sensor) with 57% of identity and 71% of similarity to ScSnf3 , and 46% of identity and 63% of similarity to ScRgt2 (Polupanov et al., 2012).PpGss1 also reveals high level of homology to Hxs1 protein of H. polymorpha (62% of identity and 77% of similarity).PpGss1 revealed lower ho mology level to HpGcr1 protein with 42% of iden tity and 60% of similarity.like the S. cerevisiae sensors, PpGss1 possesses 12 transmembrane do mains, a long Cterminal extension, which is the major distinguishing characteristic for glucose sen sors (Özcan et al., 1998), but lacks of nterminal peptide (52 amino acids) present in S. cerevisiae homologs (fig.5; fig. 2

from Polupanov et al., 2012).
The strain with knock out of the gene GSS1 has been constructed.The correct integration of deletion cassette into the genome of the Δgss1 knockout strain was verified by Southern blot and PCR.In contrast to the wildtype cells, the strain without the GSS1 gene had impaired growth for  Alcohol oxidase (AoX) replica plate overlay assay was used as preliminary examination of mi cropexophagy in Δgss1 mutant.P. pastoris wild type, Δgss1 and pep4 prb1 strains with defect of vacuolar proteinases were grown on methanol min imal medium for 2 days and then they were rep lica plated to glucose minimal medium to induce micropexophagy.Residual alcohol oxidase activity led to the redcolored cells on the plates with glu cose indicating impairment of peroxisome degra dation (Sibirny and Titorenko, 1986;Stasyk et al., 2008a).The cells of Δgss1 mutant strain, similarly to that of pep4 prb1 mutant, showed residual alco hol oxidase activity suggesting the block of pex ophagy, unlike the wildtype strain with normal inactivation of the enzyme (fig.6 from Polupanov et al., 2012).These results support the hypothesis that Gss1 is important for micropexo phagy in the methylotrophic yeast P. pastoris (Polupanov et al., 2012).In other experiment, protein samples for Western blot analysis were prepared from the cells of P. pastoris wildtype, Δgss1 and pep4 prb1 strains cultivated in methanol medium and transferred to glucose medium.for monitoring pexophagy ki netics, antibodies against P. pastoris alcohol oxi dase were used.In the wildtype strain, level of alcohol oxidase decreased during the adaptation of the cells to glucose.Unlike the wildtype, Δgss1 mutant maintained the stable alcohol oxidase level up to 9 h of glucose adaptation.Contrary to that the wildtype cells showed no detectable alcohol oxidase band.Similarly, defect I pexophagy was observed in Δgss1 mutant after peroxisome induc tion with oleate instead of methanol.To validate Data showed thus that in the cells of Δgss1 mutant peroxisomes degrade via micropexophagy but much slower than in the wildtype cells.Thus, the gene GSS1 seemed to be important for micropexo phagy (Polupanov et al., 2012).During incubation of methanolgrown cells in ethanol medium, cells of Δgss1 mutant showed drop in the amount of alco hol oxidase protein, however, the process was slow er than in the wildtype cells.Thus, gene GSS1 is only partially involved in macropexophagy.fluo rescent observations supported this conclusion.It was also shown that GSS1 is not involved in Cvt pathway and general (nonspecific) autophagy (Po lupanov et al., 2012).It was found that that deletion of 150 resi dues of Gss1 leads to the alteration of phenotype, still maintaining signaling function of Gss1.At the same time, the substitution of one conserved amino acid R180K of Gss1 protein has no visible phenotype, in contrast to corresponding changes in glucose sensors from other yeast species.It has been suggested that Cterminal cytoplasmic ex tension of PpGss1 plays different role compared to that of its homologs in Saccharomyces cerevisiae and Hansenula polymorpha (A.Polupanov and A. Sibirny, in press).Thus, the mechanism and amino acid residues responsible for glucose sens ing by Gss1 protein remain to be elucidated in the future studies.
Thus, the specific homolog of glucose trans porters, transceptor sensor Gss1 has been identified involved in glucose sensing for micropexophagy.It is also involved in glucose catabolite repression.
Low molecular-weight effector which triggers glucose signal for pexophagy.It is not known at the moment, which metabolite is the immediate signaling molecular initiating pexophagy signaling in glucose medium.It could be glucose or its me tabolite.The observation that enzymatically inac tive phosphofructokinase restored micropexophagy in glucose medium without restoration of the growth on glucose, suggests that such metabolite has to be upstream of fructose1,6bisphosphate (yuan et al., 1997;Dunn et al., 2005).The study of other mutants defective in particular steps of glycolysis could help in indentification of the im mediate effector of pexophagy in glucose medium.
Glucose signaling for pexophagy.Mechanisms of glucose signaling in S. cerevisiae have been studied in detail (Santangelo, 2006;Gancedo 2008;RubioTexeira et al., 2010).The scheme of glucose signaling during pexo phagy in this species was pro vided before (nazarko et al., 2008b).our knowled ge is quite restrictive regarding glucose signaling during pexophagy in methylotrophic yeasts.The study of thiolase and bifunctional enzyme fox3 degradation as a peroxisomal markers showed that the Slt2 (Mpk1) mitogenactivated protein kinase (MAPK) is necessary for pexophagy but not for pexophagosome formation or other nonselec tive and selective forms of autophagy.It was also showed that several upstream components of its signal transduction pathway (Pkc1, Bkc1, Mkk1 and Mkk2) are also involved in glucose signaling (Manjithaya et al., 2010).MAPK Slt2 does not participate in Cvt pathway and general (nonspe cific) autophagy.
It was proposed that pexophagy requires the simultaneous activation of this MAPK pathway and a hexosesensing mechanism acting through protein kinase A and cyclic adenosine monophos phate.Data, showing that orthologs of S. cerevisiae Mig1 and Mig2 are not apparently involved in glucose catabolite repression (Stasyk et al., 2007), suggest on possible strong differences in mecha nisms of glucose signaling between baker's and methylotrophic yeasts.
The only work on studying glucose signaling in pexophagy in methylotrophic yeasts was pub lished in the above mentioned article on the role of the αsubunite of phosphofructokinase in micro pexophagy (and not in macropexophagy) in P. pastoris (yuan et al., 1997).other components of the signaling cascade remain to be elucidated in the future research.

ethanol sensing for pexophagy in methylotrophic yeasts
Ethanol signaling for pexophagy apparently exists only in methylotrophic yeasts as in other yeast species used for pexophagy studies (S. cerevisiae, y. lipolytica).Ethanol does not induce pexo phagy of oleateinduced peroxisomes.However, practically nothing is known on ethanol sensing in yeasts, including in S. cerevisiae.nevertheless, there have to be several quite specific mechanisms of ethanol sensing and signaling.It is known that ethanol specifically and strongly induces several proteins in S. cere visiae, glucokinase being induced near 25 fold (Herrero et al., 1999).In S. cerevisiae, ethanol represses PDC1 coding for pyruvate decar boxylase through ERA regulatory sequence (liesen et al., 1996) and in Kluyveromyces lactis ethanol specifically represses the expression of ADH3 coding for mitochondrial alcohol dehydrogenase (Saliola et al., 2007).In methylotrophic yeasts, ethanol specifically activates the repression of syn thesis of the enzymes involved in methanol me tabolism in addition to pexophagy (Sibirny et al., 1989).We do not know if there are specific ethanol sensors in cytoplasmic membrane, till now no such protein was reported.Possibly ethanol is sensed by some intracellular specific sensors and/or ethanol metabolizing enzymes.There are ecr1 and adh1 mutants of the methylotrophic yeast Pichia methanolica (Pichia pinus MH4) known in which ethanol is unable to repress synthesis of the peroxisomal enzymes involved in methanol catabolism (Sibirny et al., 1987;Sibirny et al., 1991).In adh1 mutants, ethanol and methanol are utilized simultaneously and hybrid peroxisomes are produced which ap parently maintain enzymes for both methanol and ethanol metabolism, whereas in ecr1 mutants methanol is first utilized from the mixture of both alcohols.Though the genes were not isolated, adh1 mutation apparently tagged one of alcohol dehy drogenases whereas ECr1 gene possibly encodes protein involved in ethanol sensing.
In P. methanolica, attempts were made to identify a derivative of ethanol initiating pexophagy in ethanol medium.Mutants defective in distinct steps of ethanol utilization have been isolated (Tol storukov et al., 1989;Sibirny et al., 1990).It was found that pexophagy was affected in mutants icl1 defective in isocitrate lyase suggesting that isoci trate is immediate ethanol metabolite initiating pexophagy.
Thus, the mechanisms of sensing and signa ling in glucose and ethanolinduced pexophagy in yeast, in general, and methylotrophic yeasts, in particular, are far from understanding.At this mo ment, we do not know exact glucose sensors and components of signal transmitting to pexophagy machinery.In the case of ethanolinduced pexo phagy, our knowledge is at the initial stage.It could be envisaged that studies in this field will be more active in the nearest future and we will have soon the mechanistical picture of pexophagy sensing and signaling by glucose and ethanol in methylotrophic yeasts.