Atg35, a micropexophagy-specific protein that regulates micropexophagic apparatus formation in Pichia pastoris

Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAS), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. However, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35Δ mutant was not affected in macropexophagy, cytoplasm-to-vacuole targeting or general autophagy. However, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MIPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral ER to the nuclear envelope was required to organize a peri-nuclear structure (PNS), the site of Atg35 colocalization during micropexophagy. In summary, Atg35 is a new Atg protein that relocates to the PNS and specifically regulates MIPA formation during micropexophagy.Key words: Atg protein, peroxisome, micropexophagy, MIPA, nucleus     

GSH2, a gene encoding gamma-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorpha

The GSH2 gene, encoding Hansenula polymorpha gamma-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121.     

Atg28, a novel coiled-coil protein involved in autophagic degradation of peroxisomes in the methylotrophic yeast Pichia pastoris

In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy. We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris. This yeast exhibits two morphologically distinct pexophagy pathways, micro- and macropexophagy, induced by glucose or ethanol, respectively. Deficiency in ATG28 impairs both pexophagic mechanisms but not general (bulk turnover) autophagy, a degradation pathway in yeast triggered by nitrogen starvation. It is known that the micro-, macropexophagy, and general autophagy machineries are distinct but share some molecular components. The identification of ATG28 suggests that pexophagy may involve species-specific components, since this gene appears to have only weak homologues in other yeasts.     

Autophagy-related pathways and specific role of sterol glucoside in yeasts

Recently, we showed that the requirement of sterol glucoside (SG) during pexophagy in yeasts is dependent on the species and the nature of peroxisome inducers. Atg26, the enzyme that converts sterol to SG, is essential for degradation of very large methanol-induced peroxisomes, but only partly required for degradation of smaller-sized oleate- and amine-induced peroxisomes in Pichia pastoris. Moreover, oleate- and amine-induced peroxisomes of another yeast, Yarrowia lipolytica, are degraded by an Atg26-independent mechanism. The same is true for degradation of oleate-induced peroxisomes in Saccharomyces cerevisiae. Here, we review our findings on the specificity of Atg26 function in pexophagy and extend our observations to the role of SG in the cytoplasm to vacuole targeting (Cvt) pathway and bulk autophagy. The results presented here and elsewhere indicate that Atg26 might increase the efficacy of all autophagy-related pathways in P. pastoris, but not in other yeasts. Recently, it was shown that P. pastoris Atg26 (PpAtg26) is required for elongation of the pre-autophagosomal structure (PAS) into the micropexophagic membrane apparatus (MIPA) during micropexophagy. Therefore, we speculate that SG might facilitate elongation of any double membrane from the PAS and this enhancer function of SG becomes essential when extremely large double membranes are formed.     

Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica

Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.     

Cloning of the GSH1 and GSH2 genes complementing the defective biosynthesis of glutathione in the methylotrophic yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the gamma-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh- phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24 with the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme gamma-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Differences in glucose sensing and signaling for pexophagy between the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris

The mechanism(s) of glucose sensing for inducing the autophagic peroxisome degradation (pexophagy) is not known. Recently, we have found that defects in the S. cerevisiae PKA-cAMP signaling pathway due to knockouts of GPR1 and/or GPA2 suppressed glucose-induced degradation of peroxisomal thiolase. Here we report that single defects of high (SNF3) and low (RGT2) affinity glucose sensors involved in glucose-dependent induction of hexose transporters have only a slight effect on glucose-induced degradation of peroxisomal thiolase, although simultaneous defects of both sensors, SNF3 and RGT2 (which are known to strongly affect glucose transport) strongly inhibit this process in S. cerevisiae. Most likely, glucose is sensed for pexophagy using the Gpr1 sensor involved in the PKA-cAMP signaling pathway. In the methylotrophic yeast P. pastoris, however, knock out of S. cerevisiae orthologs of GPR1 and GPA2 did not affect glucose-induced degradation of oleate-induced thiolase or the methanolinduced key peroxisomal protein, alcohol oxidase. This implies that glucose sensing for pexophagy is different in baker's and methylotrophic yeasts.     

Paraphyly of organelle DNAs in Cycas Sect. Asiorientale s due to ancient ancestral polymorphisms

Background  

This study addresses the apportionment of genetic diversity between Cycas revoluta and C. taitungensis, species that constitute the section Asiorientales and represent a unique, basal lineage of the Laurasian genus Cycas. Fossil evidence indicates divergence of the section from the rest of Cycas at least 30 million years ago. Geographically, C. taitungensis is limited to Taiwan whereas C. revoluta is found in the Ryukyu Archipelago and on mainland China.