The role of Hansenula polymorpha MIG1 homologues in catabolite repression and pexophagy

In the methanol-utilizing yeast Hansenula polymorpha , glucose and ethanol trigger the repression of peroxisomal enzymes at the transcriptional level, and rapid and selective degradation of methanol-induced peroxisomes by means of a process termed pexophagy. In this report we demonstrate that deﬁciency in the putative H. polymorpha homologues of transcriptional repressors Mig1 ( HpMig1 and HpMig2 ), as well as HpTup1, partially and differentially affects the repression of peroxisomal alcohol oxidase by sugars and ethanol. As reported earlier, deﬁciency in HpTup1 leads to impairment of glucose- or ethanol-induced macropexophagy. In H. polymorpha mig1mig2 double-deletion cells, macropexophagy was also substantially impaired, whereas micropexophagy became a dominant mode of autophagic degradation. Our ﬁndings suggest that homologues of the elements of the Saccharomyces cerevisiae main repression pathway have pleiotropic functions in H. polymorpha .


Introduction
It is well established that intact peroxisomes are indispensable for methylotrophic growth in yeasts. These organelles harbour key enzymes that catalyze the first steps of methanol utilization [as alcohol oxidase (AO), catalase, dihydroxyacetone synthase], and provide compartmentalization of toxic methanol catabolites, formaldehyde and hydrogen peroxide (Veenhuis et al., 1983b;van der Klei et al., 2006). Peroxisomes, however, are redundant for growth on rich carbon sources such as hexoses and disaccharides. When methanolgrown cells are shifted to sugar carbon substrates or ethanol, peroxisomes are rapidly and selectively degraded in vacuoles (Veenhuis et al., 1983a), whereas genes of peroxisomal enzymes are subjected to tight transcriptional repression (Roggenkamp et al., 1984). Such coordinated genetically programmed catabolite regulation ensures a fast and efficient cellular response to environmental changes.
It was originally assumed that the molecular pathway of glucose catabolite repression in methylotrophic yeasts may mimic that of the conventional yeast Saccharomyces cerevisiae (the so-called Snf1-Mig1 pathway). Its main components, the lack of which abolishes repression signalling, are hexokinase II subunit, Snf1 protein kinase, and the Mig1 repressor that tethers or facilitates the binding of the Tup1-Ssn6 general repressor to promoters of repressible genes in the presence of glucose (Gancedo, 1998;Papamichos-Chronakis et al., 2004). Several observations support this hypothesis. First, Mig1-Ssn6-Tup1 and the Snf1 complexes are highly conserved among different yeasts (Gancedo, 1998). Second, promoters of H. polymorpha alcohol oxidase (Pereira & Hollenberg, 1996) and maltase (Alamae et al., 2003) genes were demonstrated to be repressed by glucose in S. cerevisiae, suggesting that they are targets of the host's repression pathway. Recently, hexose phosphorylation activity was shown to be essential for repression signalling in H. polymorpha: hexokinase mutant was insensitive to fructose (but not glucose) repression, whereas in double hexo-and glucokinase mutant, glucose repression was also abrogated (Kramarenko et al., 2000). The above findings suggested that glucose-repressible genes of C-1 metabolism in H. polymorpha may be the target of Mig1/(Tup1-Ssn6)mediated transcriptional repression, analogous to the case of baker's yeast. However, Oliveira et al. (2003) reported that HpTup1 is not essential for glucose repression of peroxisomal enzymes in H. polymorpha.
Adding to the emerging complexity of repression pathway in this yeast, we recently identified a hexose transporter homologue, HpGcr1, which is most probably involved in the first stages of glucose (but not of sucrose or ethanol) signalling for repression, but may also interfere with highaffinity glucose transport (Stasyk et al., 2004). Its close homologues in S. cerevisiae, the putative glucose sensors Snf3 and Rgt2, were not implicated in the repression mechanism, but instead differentially regulate the induction of functional hexose transporters in response to extracellular glucose (Johnston & Kim, 2005). It has to be emphasized that, in H. polymorpha and other methylotrophs, disaccharides and ethanol are potent repressors of gene expression involved in C-1 metabolism (Sibirny et al., 1988;Stasyk et al., 2004). However, nothing is known of the specific molecular components of ethanol-or disaccharide-triggered repression, whether different carbon source-dependent pathways physically converge, and, if so, at what stage they do so.
With regard to pexophagy, significant progress has been achieved in recent years in understanding its molecular mechanisms and regulation, with H. polymorpha being a very productive model organism, as detailed in recent reviews (Dunn et al., 2005;Monastyrska & Klionsky, 2006;Sakai et al., 2006).
Two morphologically distinguishable pexophagy mechanisms have been observed in H. polymorpha: selective macropexophagy-triggered by a shift in carbon source (i.e. from methanol to glucose or ethanol) (Veenhuis et al., 1983a;Tuttle et al., 1993), or cold shock ; and nonselective micropexophagy triggered upon nitrogen starvation, leading to bulk turnover by general autophagy (Monastyrska et al., 2002). Most ATG (AuTop-haGy-related) genes involved in pexophagy identified so far in H. polymorpha and other yeasts are conserved among eukaryotes and also appear to affect general autophagy. Those essential only for pexophagy are in many cases organism-specific (Meijer et al., 2007). The latter include H. polymorpha ATG11 and ATG25 (Monastyrska et al., 2005), ATG26 (our unpublished results), and TUP1 (Leao-Helder et al., 2004). HpTup1 is a first representative of transcriptional repressors involved in pexophagy. As macropexophagy, in contrast to nitrogen starvation-induced microautophagy, is insensitive to cycloheximide (CHX) (i.e. independent of protein synthesis de novo) (Monastyrska et al., 2005), the function of HpTup1 in pexophagy has been suggested to be indirect (Leao-Helder et al., 2004).
Knowledge of the molecular mechanisms of catabolite regulation in H. polymorpha is of basic and practical interest. Mutants with altered catabolite regulation of the AO gene promoter may be promising hosts for the expression of recombinant proteins in methanol-free media (Krasovska et al., 2007), and protein products can be targetted to peroxisomes applying this organelle as a storage vesicle (van Dijk et al., 2000). The availability of the full H. polymorpha genome sequence (Ramezani-Rad et al., 2003) made it possible to search for orthologues of S. cerevisiae genes involved in catabolite repression and to study their function. In this report we analyse the possible role of two H. polymorpha Mig1 homologues (designated HpMig1 and HpMig2) in the processes of catabolite repression and pexophagy.
The Escherichia coli DH5a strain was used as a host for propagation of plasmids and grown at 37 1C in Luria-Bertani (LB) medium as described in Sambrook et al. (1989).

Molecular-biology techniques
Standard cloning and DNA manipulation techniques were applied (Sambrook et al., 1989). Genomic DNA of H. polymorpha was isolated using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Restriction endonucleases and DNA ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer's specifications. PCR-amplification of the fragments of interest was performed with Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) according to the manufacturer's specification. The primers used in this study are listed in Table 1. The transformation of H. polymorpha by electroporation was carried out as described previously (Faber et al., 1994).

Protein sequence analyses
For analysis of DNA and amino acid sequences, MACVECTOR software (IBI, New Haven, CT) was used. Sequence alignments and phylogenetic analysis were performed using the CLUSTALW version 1.6 algorithm (Corpet, 1988). The BLAST Network Service of the National Center for Biotechnology Information (Bethesda, MD; http://www.ncbi.nlm.nih.gov/ BLAST/) was used to search for amino acid sequence similarities. For pattern and profile search, the PROSCAN program at http://npsa-pbil.ibcp.fr/cgibin/pattern_prosite.pl was used (Combet et al., 2000).

Construction of HpMIG1 and HpMIG2 deletion strains
A HpMIG1 (GenBank accession number EF523343) deletion cassette was constructed as follows: a 0.93-kb fragment containing sequences of the 3 0 -flanking region of HpMIG1 was amplified by PCR using genomic DNA of H. polymorpha as a template and the primers Mi12Cf and Mi12Cr carrying restriction sites for EcoRI and BamHI (Table 1). The 3 0 -flanking fragment was inserted into the EcoRI/BamHIdigested plasmid pBSKT (Lombo et al., 1997). Next, a 0.99-kb fragment consisting of the 5 0 -flanking region of HpMIG1 was amplified by PCR with the primers Mi12Nf and Mi12Nr (Table 1) carrying restriction sites for HindIII and NotI, respectively. The 5 0 -flanking PCR fragment of HpMIG1 and BamHI/NotI-digested HpLEU2 fragment were cloned into the BamHI/NotI-digested plasmid pBSKT-3 0 MIG1, thus generating a deletion cassette for HpMIG1 (plasmid p19-mig12::HpLEU2). The derivative plasmid pOH7 was constructed by replacing the PstI fragment of HpLEU2 on p19-mig12::HpLEU2 with the PstI fragment harbouring the APH gene expressed under the constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase from plasmid pGLG578 (Sohn et al., 1999) (Fig. 1).
A HpMIG2 (GenBank accession number EF523344) deletion cassette was constructed as follows: a 1.97-kb fragment containing sequences of the 3 0 -flanking region of HpMIG2 was amplified by PCR using genomic DNA of H. polymorpha NCYC495 leu1-1 as a template and the primers OL155 and OL156 carrying restriction sites for HindIII (Table 1, Fig. 1). The 3 0 -flanking fragment was inserted into the HindIIIdigested plasmid pYT1 (Tan et al., 1995), carrying the ScLEU2 gene as a selection marker, to create pYT1-3 0 MIG2. Next, a 1.39-kb fragment consisting of the 5 0 -flanking region of HpMIG2 was amplified by PCR with the primers OL153 and OL154 carrying restriction sites for BamHI and XbaI, respectively. The 5 0 -flanking PCR fragment of HpMIG2 was cloned into the BamHI/XbaI-digested plasmid pYT1-3 0 MIG2, to create pOH8, generating a deletion cassette for HpMIG2.
The plasmid pOH8 was used as a template for PCR with primers OL153 and OL156, producing a 5.56-kb fragment comprising ScLEU2 flanked by MIG2 5 0 -and 3 0 -sequences. The deletion cassette was transformed into NCYC495leu1-1, and prototrophic transformants selected.
Total genomic DNA isolated from potential Dmig1 knock-out strains (p19-mig12::HpLEU2 and pOH7 transformants) was used as a template for PCR and Southern blot analyses. Primer pairs OL54/OL55, OL54/OL57 and OL54/ OL143 (Table 1, Fig. 1) were used to determine the presence Mi12Nf Mi12Nr Mi12Cf Mi12Cr of corresponding knock-out cassettes in Dmig1 and Dmig1 leu1-1 genomic DNA. PCR analysis (data not shown) indicated the presence of expected bands in the tested strains: a 0.45-kb band generated by OL54/OL55, a 2.24-kb band (OL54/OL57), and the absence of the band (OL54/ OL143) in the control strain NCYC495leu1-1. The 2.91-kb band (for Dmig1) and 3.9-kb band (for Dmig1leu1-1) generated by OL54/OL57 and the 0.5-kb band generated by OL54/OL143 were present in knock-out strains. The pair of primers OL54/OL55 did not generate a band in the Dmig1 and Dmig1 leu1-1strains. Primer pairs OL91/OL92 and CK15/OL156 (Table 1, Fig. 1) were used for PCR analysis of the Dmig2 strain. The 1.2-kb band generated by OL91/OL92 from genomic DNA of NCYC495leu1-1 was absent in the deletion strain. The pair of primers CK15/OL156 was used for indication of a correctly targetted chromosomal integration of mig2:: HpLEU2 fragment. The 2.5-kb band was obtained with PCR for Dmig2 and was absent in the control strain NCYC495 leu1-1. Proper integration of the single HpMIG1 and HpMIG2 disruption cassettes was confirmed by Southern blot analysis (data not shown).
The identified Dmig1 was used in all biochemical and physiological experiments in this study. Dmig1 leu1-1 was used for construction of the Dmig1Dmig2 double-deletion mutant. The Dmigl leu1-1 and Dmig2 strains were crossed and diploid hybrids were selected on YNS medium supplemented with G418 (1.0 g L À1 ) without leucine. After sporulation, the Dmig1Dmig2 double-deletion mutant was selected among spore progeny on the same medium, and confirmed for the presence of HpMIG1 and HpMIG2 deletion cassettes as detailed above for single-deletion mutants. Mating and sporulation techniques were performed using established procedures (Gleeson & Sudbery, 1988).

Biochemical and ultrastructural analyses
Preparation of crude cell extracts, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analyses and preparation of cells for electron microscopy and immunocytochemistry were carried out as described in Waterham et al. (1994).

Results
Identification of H. polymorpha homologues of the S. cerevisiae Mig1 transcriptional repressor A search in the H. polymorpha genome database (Ramezani-Rad et al., 2003) revealed the presence of two putative homologues of S. cerevisiae Mig1 (ScMig1) C2H2 zinc-finger (ZnF) transcriptional repressor (Nehlin & Ronne, 1990). Similar results were obtained after probing the genome database with full-length ScMig1, or its N-terminal ZnF DNA-binding domain, which is highly conserved in all yeast Mig1 homologues (Ostling et al., 1996;Cassart et al., 1997;Zaragoza et al., 2000;Carmona et al., 2002), or the full-length sequence of the related S. cerevisiae Mig2 protein (Lutfiyya & Johnston, 1996). As expected, the two identified H. polymorpha Mig1 homologues, designated as HpMig1 and HpMig2, exhibit limited overall homology to other yeast Mig proteins: for instance, 26% identity and 41% similarity to ScMig1; 32% identity and 43% similarity to Candida albicans Mig1 (Zaragoza et al., 2000); and 34% identity and 45% similarity to Schwanniomyces occidentalis Mig1 (Carmona et al., 2002). However, in the region of N-terminal ZnF domains, the similarity is high: 80% and 76% identity to ScMig1 for HpMig1 and HpMig2, respectively (Fig. 2a). Other predicted H. polymorpha proteins from the genome database exhibit o 50% identity in the ZnF region to ScMig1.
HpMig1 and HpMig2 differ in length (480 and 402 amino acid residues, respectively) and are 33% identical and 44% similar to each other. Both H. polymorpha proteins exhibit o 69% identity in their ZnF domains to ScMig2, and o 66% to the related S. cerevisiae Yer028c (Mig3) (Lutfiyya et al., 1998). However, the DNA-binding domains of both H. polymorpha Mig proteins are the most similar (4 85% identity) to the corresponding regions of Mig-like CreA repressors from filamentous fungi (Ronne, 1995).
Despite the low sequence conservation aside the ZnF region, yeast Mig homologues possess other semi-conserved regions (Cassart et al., 1997), present also in HpMig1 and HpMig2 ( Fig. 2b and c), i.e. the so-called C-terminal 'effector domain' (Ostling et al., 1996). In addition, the H. polymorpha proteins harbour an amino acid region highly similar to fungal CreA repressors that is absent in many yeast Mig proteins (Fig. 2d). They also possess consensus phosphorylation sites for cAMP-dependent protein kinase, and Snf1 protein kinase (not shown), and therefore may potentially be the subject of corresponding regulation (Treitel et al., 1998). Taken together, our analyses suggest that the identified HpMig1 and HpMig2 most probably represent potential ScMig1 orthologues of this methylotrophic yeast. The corresponding sequences were deposited in GenBank under accession numbers EF523343 (HpMIG1), and EF523344 (HpMIG2).
HpMig1 and HpMig2 may have a redundant function in transcriptional regulation as observed in S. cerevisiae (Lutfiyya et al., 1998), a double-deletion Dmig1Dmig2 mutant strain was isolated from spore progeny of the diploid, resulting from crossing Dmig1 and Dmig2 null mutants.
We observed that Dmig1 and Dmig2 mutants did not differ from the wild-type strain in growth on solid media supplemented with various carbon sources, namely glucose, sucrose (glycolytic substrates), ethanol and methanol, whereas the double-deletion mutant exhibited retarded growth on sugar substrates (Fig. 3a). The mutants behaved similarly on plates with low sugar concentrations (0.1% w/v) (not shown). However, Dmig1 and Dmig1Dmig2 mutants could grow on methanol plates in the presence of 2deoxyglucose, suggesting an impairment of glucose repression. The H. polymorpha Dtup1 mutant, which is not disturbed for glucose repression (Oliveira et al., 2003), was used as a control.
Detailed analysis of growth kinetics in liquid cultures confirmed that mig mutants exhibit a wild-type growth rate on methanol and ethanol. When grown on sugar substrates, the lag phase in Dmig1 and double Dmig1Dmig2 mutants was extended, but growth was not affected in the exponential phase (Fig. 3b). This observation suggests that HpMig1 and HpMig2 may be involved in a transient transcriptional response upon glucose adaptation.

Assessment of the role of HpMig1, HpMig2 and HpTup1 in catabolite repression
To gain further insight into HpMig1, HpMig2 and HpTup1 function in repression triggered by rich carbon sources, we assayed AO protein levels in the mutants. A very moderate impairment of AO repression was observed for Dmig1, Dmig1Dmig2 and Dtup1 mutants incubated with low (0.1%) or high (1%) concentrations of glucose (Fig. 4). Pre- Fig. 3. Growth properties of Hansenula polymorpha mig mutants on various carbon sources. (a) Equal volumes of YPS pre-grown cell suspensions were loaded in three dilutions on agarized media. Carbon substrate abbreviations are: Mth, methanol; Glc, glucose; 2-DOG, 2deoxyglucose; Sucr, sucrose; Eth, ethanol. Cells were incubated for 1 day on sugar substrates, 2 days on methanol or ethanol, and 3 days on 2-DOG-containing medium. (b) Kinetics of growth in the liquid culture. Cells of the indicated H. polymorpha strains were pregrown on rich YPS medium and shifted to the mineral media with different carbon sources. OD 600 nm was measured at the indicated time points after the shift.
viously described by us Dgcr1 mutant strongly affected in glucose repression and constitutively synthesizing AO in glucose medium (Stasyk et al., 2004) served as a positive control. Interestingly, when methanol was added to glucose medium, the defect in repression in Dmig1 and Dmig1Dmig2, but not in Dtup1, became more pronounced (Fig. 4). This suggests that certain transcriptional induction components are induced for AO synthesis in mig1 cells and may explain the ability of mutants to grow on methanol in the presence of 2-deoxyglucose (Fig. 3A). However, the AO protein level in the double Dmig1Dmig2 mutant on carbon source mixture was considerably lower relative to glucosegrown Dgcr1 cells or methanol-grown wild-type cells (Fig. 4  and legend).
Similarly, AO synthesis in Dmig1, Dmig2, Dmig1Dmig2, and Dtup1 cells was only weakly derepressed in cells exposed to sucrose or ethanol (Fig. 4), suggesting a limited effect of the analysed mutations on the repression pathway(s) in H. polymorpha. We excluded peroxisome degradation as a principal factor influencing AO protein level in the analysed mutants grown on multicarbon substrates, as pexophagy is blocked (in tup1) or impaired in these strains (see next section).

Hansenula polymorpha Mig1 homologues interfere with pexophagy
Hansenula polymorpha HpTup1 is the only transcriptional repressor shown so far to be essential for macropexophagy (Leao-Helder et al., 2004). We were interested in elucidating whether its possible partners in transcriptional regulation, HpMig1 and HpMig2, also affect this process. In AO activity plate assays, upon a shift of methanol-grown cells to glucose or ethanol, we observed that Dmig1 and Dmig1Dmig2, but not Dmig2, cells exhibit enhanced residual AO activities relative to wild-type controls. However, the defect in pexophagy in Dmig1 was less pronounced relative to Dtup1 cells (not shown). Similarly to the case for Dtup1, no mig mutant was significantly affected in general autophagy upon nitrogen starvation, as judged by means of the Phloxine-B plate test (Tsukada & Ohsumi, 1993) (not shown).
Detailed time-course analysis of pexophagy by following AO protein level revealed that the rate of AO degradation differed between the wild-type strain and mig mutants when methanol-grown cells were exposed to glucose or ethanol (Fig. 5a). However, Western blots demonstrated that, also during the growth of Dmig1Dmig2 cells on methanol, minor AO degradation bands could be observed that were absent in wild-type controls (Fig. 5b), indicating that peroxisomes are degraded and that pexophagy is not completely blocked under these conditions in Dmig1Dmig2 cells.
Electron microscopy of methanol-grown Dmig1Dmig2 cells exposed to ethanol revealed that peroxisomes indeed undergo degradation and that AO protein can be detected in vacuolar lumen (Fig. 6c). Unexpectedly, whereas in wildtype controls organelles were degraded via macropexophagy (not shown), in Dmig1Dmig2 cells the sequestration of organelles to be degraded was rarely observed (Fig. 6b). Instead, the morphological characteristics of micropexophagy, i.e. vacuolar protrusions surrounding peroxisome clusters, were prominent (Fig. 6a).

Discussion
This paper describes the role of H. polymorpha transcriptional repressors in catabolite repression and peroxisome degradation (catabolite inactivation) induced by rich carbon sources. We demonstrated that the repression pathway for peroxisomal AO in this yeast apparently only partially relies on the two Mig1 homologues, irrespective of the carbon source (glucose, sucrose or ethanol). However, addition of the inducer methanol elevates the AO protein level in Dmig1Dmig2 mutant incubated with glucose ( Fig.  4), suggesting that this mutant exhibits a so-called 'inducible' phenotype (Sibirny et al., 1988).
In S. cerevisiae, Mig1 is regulated by glucose (Ostling & Ronne, 1998) and facilitates binding of the general repressor complex Tup1-Ssn6 to the promoters of glucose-repressible genes (Gancedo, 1998). However, Tup1 is involved in pleiotropic functions through interaction with specific DNA-binding proteins for each functionally related set of genes (reviewed in Malave & Dent, 2006), while the Mig1dependent regulation is thought to be more specific, with a limited set of the target genes that include those repressed by glucose (Murad et al., 2001). Therefore, our results are in line with the previous observation that HpTup1 is not required for glucose repression of peroxisomal enzymes (Oliveira et al., 2003), and suggest that, at least for alcohol oxidase, operation of the classical S. cerevisiae pathway with Mig1/2-mediated Tup1-Ssn6 binding to the repressible promoter is unlikely. However, the redundant function of these repressors cannot be excluded, and the effect of combining all three mutations in one strain would be interesting to examine. An alternative explanation is the involvement of other still-unknown repressor(s). Therefore, further studies are required to obtain conclusive results on   6. Hansenula polymorpha Dmig1Dmig2 cells are disturbed in macropexophagy but not in microautophagy. Methanol-grown Dmig1Dmig2 cells were exposed to ethanol for 1 h. (a) Peroxisomes are surrounded by vacuolar protrusions, indicative of microautophagy (arrows). (b) A peroxisome is sequestered by additional membrane structures. However, sequestration is not completed (arrow). (c) Immunolabelling reveals that, in methanol-grown Dmig1Dmig2 cells exposed to ethanol, AO protein is localized both to peroxisomes and to the vacuole, suggesting functional organelle degradation (arrows). M, mitochondrion; V, vacuole; P, peroxisome; N, nucleus. The bar represents 1 mm.
whether the effect of mig1/2 and tup1 mutations on catabolite repression in response to different carbon sources is direct or indirect. It also has to be investigated whether the analysed mutations interfere with the regulation of other glucose-repressible genes in H. polymorpha, for instance maltase, the regulation of which has been suggested to be Mig-dependent (Alamae et al., 2003).
The pleiotropic function of Tup1 in yeast was recently extended to regulation of macropexophagy (Leao-Helder et al., 2004). The finding that micropexophagy in H. polymorpha (Monastyrska et al., 2005) and P. pastoris (Tuttle & Dunn, 1995) is sensitive to cycloheximide suggests that it is dependent on protein synthesis, and, consequently, on transcriptional regulation.
Our data suggest that the effect of Tup1 deletion on pexophagy is not mimicked by a similar deficiency in HpMig homologues. In H. polymorpha tup1 mutant, macropexophagy is completely blocked, whereas micropexophagy occurs in the wild-type manner, i.e. only upon nitrogen starvation but not upon a shift of the carbon source (Leao-Helder et al., 2004). In the Dmig1Dmig2 deletion strain, macropexophagy may also be impaired, whereas micropexophagy seems to be constitutively induced. This resembles the phenotype of H. polymorpha Datg25 mutant, which also displayed constitutive microautophagy in conjunction with a block in macropexophagy (Monastyrska et al., 2005). This apparent similarity of the phenotypes of H. polymorpha Dmig1Dmig2 and Datg25 mutants suggests that certain macropexophagy-specific components may be down-regulated in the absence of Mig repressors, and, hence, Mig repressors may be positive regulators of macropexophagy. Further research to elucidate the details of these effects is required, including the use of microarray approaches.
With regard to the control of micropexophagy, Mig1 homologues and Tup1 seem to have distinct roles depending on environmental conditions. Mig1 proteins may be negative regulators of micropexophagy, whereas Tup1 acts as a positive regulator, but only upon a shift in the carbon source and not upon nitrogen depletion conditions. A tentative model explaining such regulations is presented in Fig. 7. Fig. 7. Tentative model of the involvement of HpMig-and HpTup1-mediated transcriptional repression in the regulation of pexophagy. We suggest that certain components of macropexophagy-specific machinery (such as HpAtg25) may be negative regulators of micropexophagy. Mig1/2 and Tup1 are indirect positive regulators of such hypothetical proteins (AtgX), which are activated posttranslationally by the effectors of macropexophagy, glucose and ethanol, and inhibit the hypothetical micropexophagic component AtgY. AtgX level in the absence of either Mig1/2 or Tup1 is decreased. Nitrogen starvation confers positive regulation of AtgY by inducing its expression and repressing macropexophagy at the protein level. In the absence of Tup1, but not Mig1/2, micropexophagy occurs only upon nitrogen starvation. We suggest that AtgY may be involved in the development of microautophagic vacuoles. CHX, cycloheximide.