Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.     

PpATG9 encodes a novel membrane protein that traffics to vacuolar membranes, which sequester peroxisomes during pexophagy in Pichia pastoris

When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.     

毕赤酵母过氧化物酶体吞噬的研究进展

过氧化物酶体吞噬是生物体的一种重要自我调控方式。过氧化物酶体吞噬是多种吞噬相关蛋白的共同作用,而且吞噬相关蛋白质之间的作用具有严格的时序性。由于毕赤酵母有两种吞噬方式、全基因组序列已知、基因操作技术成熟,所以毕赤酵母是研究过氧化物酶体吞噬的良好素材,也是目前研究的热点。本文对近年来毕赤酵母过氧化物酶体吞噬的启动、两种吞噬类型的形成、过氧化物酶体在液泡中降解的研究进行梳理,为毕赤酵母过氧化物酶体吞噬的进一步研究奠定基础。     

Intracellular ATP correlates with mode of pexophagy in Pichia pastoris

The methylotrophic yeast Pichia pastoris can degrade peroxisomes selectively though two distinct pexophagic pathways, viz., micropexophagy and macropexophagy. These micro- and macropexophagy pathways are induced by adaptation of methanol-grown cells to glucose-containing and ethanol-containing media respectively. However, our understanding of the molecular signal(s) that determine which pathway is activated or repressed in response to environmental changes is limited. In this study, the determinant for these pathways was sought using cells undergoing pexophagy under a variety of conditions. Micropexophagy and macropexophagy were distinguished in living cells by fluorescence microscopy. Our results indicate that glucose and ethanol were not specific inducers of micro- and macropexophagy respectively. Micropexophagy was found to be more sensitive to ATP-depletion than macropexophagy, suggesting that the micropexophagic process requires a higher level of ATP than the macropexophagic process. From these and other results, we postulate that intracellular ATP levels play an important role in determining which pexophagic pathway is activated.     

Apg2 is a novel protein required for the cytoplasm to vacuole targeting, autophagy, and pexophagy pathways

To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions. Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway. Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole. Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut, and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways. In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes. Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are needed for vacuolar import through both the Cvt pathway and autophagy. Biochemical studies revealed that Apg2 is a peripheral membrane protein. Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.     

Early and late molecular events of glucose-induced pexophagy in Pichia pastoris require Vac8

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Gss1 protein of the methylotrophic yeast Pichia pastoris is involved in glucose sensing, pexophagy and catabolite repression

In the yeast Saccharomyces cerevisiae, the one-at-a-time deletions of either the high-affinity glucose sensor gene SNF3 or the low-affinity glucose sensor gene RGT2 only slightly reduced pexophagy; however, deleting both genes greatly reduced pexophagy, evincing interaction beyond the sum of the additive effects, as recently shown. The present study identifies the only ScSNF3/RGT2 ortholog in the methylotrophic yeast Pichia pastoris (designated as PpGSS1, from GlucoSe Sensor) and describes its roles in autophagic pathways (non-selective and selective). GSS1 knock-out strain has been constructed. The experiments support the hypothesis that Gss1 plays an important role in autophagic degradation of peroxisomes and glucose catabolite repression in P. pastoris.     

免疫法筛选酵母HSA-IL-11融合蛋白转化子

报道了一种筛选高表达融合蛋白HSA-IL-11的毕赤酵母转化子的免疫双膜筛选法.将生长在醋酸纤维素滤膜上的转化子进行原位诱导,再用硝酸纤维素滤膜对表达的蛋白进行原位捕捉,并经封闭过夜后使用抗HSA抗体进行免疫杂交,再用标记二抗进行显色.根据显色强弱将转化子分为强阳性、中等和阴性三类,再用抗IL-11抗体进行复筛验证.结...     

The membrane dynamics of pexophagy are influenced by Sar1p in Pichia pastoris

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.     

Recombinant protein expression in Pichia pastoris

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.     

Mechanisms of autophagy and pexophagy in yeasts

Autophagy is a process of recycling of the intracellular constituents using vacuoles (lysosomes). General autophagy occurs due to involvement of highly conservative components found in all eukaryotes, from yeasts to higher plants and humans. Autophagy also could be a selective process and be involved in regulation of the cellular number of organelles, including that of peroxisomes. The process of specific autophagic peroxisome degradation is known as pexophagy. Yeasts appear to be convenient model for studying molecular mechanisms of pexophagy, and most known ATG genes (from the term AuTophaGy) were identified in yeast studies. This review examines characteristics of general autophagy, other types of autophagy as well as pexophagy, in particular, functions of Atg proteins in general autophagy and in macro- and micropexophagy. Special attention is given to mechanisms of phagophore assembly, the role of phosphatidylinositol-3-phosphate in pexophagy, the role of peroxines (proteins involved in peroxisome biogenesis) in pexophagy, as well as properties of Atg proteins specifically involved in micropexophagy.     

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.     

Copper is required for prion protein-associated superoxide dismutase-I activity in Pichia pastoris

The prion protein (PrP) is the key protein implicated in transmissible spongiform encephalopathies. It is a metalloprotein that binds manganese and copper. The latter is involved in the physiological function of the protein. We have previously found that PrP expression in Pichia pastoris affects intracellular metal ion concentrations and that formation of protease-resistant PrP is induced by additional copper and/or manganese. In this study, we show that heterologously expressed PrP is post-translationally modified and transported to the cell wall. We found by combining three different test systems that PrP itself had gained superoxide dismutase-like activity in P. pastoris. However, this activity could not be inhibited by KCN and depended on additional copper in the medium. Thus, this study defines the conditions under which PrP exhibits superoxide dismutase-like activity by showing that copper must be present for the protein to participate in scavenging and detoxification of reactive oxygen species.     

利用巴斯德毕赤酵母表达外源蛋白的研究进展

随着基因工程技术的迅速发展,已有数百种外源蛋白利用巴斯德毕赤酵母表达系统获得了成功表达。本综述了该表达系统的优点、系统的构成,外源基因转化该表达系统的方式及表达特点,阐述了该系统在生产外源蛋白上的广泛应用．并重点分析了影响外源蛋白在该表达系统中表达的因素及优化策略等。     

重组人白细胞介素11在毕氏酵母中的表达

将人白细胞介素11基因选用酵母偏爱密码子人工合成全基因,克隆到酵母分泌型表达载体pGENYk中,酶切线性化后原生质体转化导入酵母细胞进行整合,G418筛选得到多拷贝转化子,甲醇诱导表达,纯化制备产物,经过SDS－PAGE、Western印迹及体内外生物学活性等分析表明,产物活性与E.coli融合表达的Neumega一致。     

人血清白蛋白-血管钠肽融合蛋白在毕赤酵母中的表达

人工合成VNP基因,通过酶连构建HSA和VNP基因的融合基因,插入表达载体pPIC9K,电转至毕赤酵母GS115,构建成工程茵,甲醇诱导表达。重组表达质粒经双酶切验证构建正确;表达产物经SDS-PAGE分析分子量为69 000 Da,与理论值相符;Western blot鉴定产物兼有HSA和VNP免疫原性,说明其为杂合分子;兔胸主动脉环离体灌流实验证明融合蛋白具有舒张血管活性。本研究说明毕赤酵母适于HSA-VNP融合蛋白的表达,为进一步开发稳定的VNP药物提供了生物制备方法。     

Expression and characterization of a synthetic protein C activator in Pichia pastoris

Protein C activators are proteases that activate protein C in the mammalian coagulation system. A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway. We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence. This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the alpha-factor signal sequence. A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth. A two-step purification scheme was devised to purify ILPCA to approximately 80% purity. The ILPCA produced has an apparent molecular weight of approximately 68 kDa and a deglycosilated molecular weight of 28 kDa. Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K(m) of 77 nM and a k(cat) of 0.39 s(-1). In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations.     

A system for dual protein expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.     

Characterization of a gene encoding a Pichia pastoris protein disulfide isomerase

Protein disulphide isomerases belong to the thioredoxin superfamily of protein-thiol oxidoreductases that have two double-cysteine redox-active sites and take part in protein folding in the endoplasmic reticulum (ER). We report here the cloning of a Pichia pastoris genomic DNA fragment (2919 bp) that encodes the full length of a protein disulphide isomerase (PpPDI). The deduced amino acid sequence of PDI consists of 517 residues and carries the two characteristic PDI-type redox-active domains -CGHC-, separated by 338 residues, and two potential N-glycosylation sites. The N-terminal end forms a putative signal sequence, and an acidic C-terminal region represents a possible calcium-binding domain. Together with the -HDEL ER retrieval sequence at the C-terminus, these features indicate that the gene encodes a redox-active ER-resident protein disulphide isomerase. The nucleotide sequence, which also contains two other open reading frames, has been submitted to the EMBL Nucleotide Sequence Database, Accession No. AJ302014.