scholarly journals Glucose-induced Autophagy of Peroxisomes in Pichia pastoris Requires a Unique E1-like Protein

1999 ◽  
Vol 10 (5) ◽  
pp. 1353-1366 ◽  
Author(s):  
Weiping Yuan ◽  
Per Eivind Strømhaug ◽  
William A. Dunn
Keyword(s):  
Pichia Pastoris ◽  
Mutant Cell ◽  
Alcohol Oxidase ◽  
Knockout Mutant ◽  
The Family ◽  
Late Event ◽  
Human Proteins ◽  
Ester Linkage ◽  
Vacuolar Membranes ◽  
In Cells

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-inducedselective autophagy),gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7gene. 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 gsa7Δ had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, aGSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog ofGSA7 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, andUBA3). When either of these sites was mutated, the resulting mutants [Gsa7(ΔATP)-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(ΔATP)-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.

scholarly journals Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases

2009 ◽  
Vol 76 (4) ◽  
pp. 1062-1070 ◽  
Author(s):  
Anna Steinle ◽  
Sabrina Witthoff ◽  
Jens P. Krause ◽  
Alexander Steinbüchel
Keyword(s):  
Pichia Pastoris ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Medium Composition ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
C Terminus ◽  
Fermentation Parameters ◽  
Optimization Of Fermentation ◽  
In Cells

ABSTRACT Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-l-arginyl-poly-l-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA 6308 and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA6308Δ1, which was truncated by one amino acid at the C terminus; point mutated CphA6308C595S; and the combined double-mutant CphA6308Δ1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA6308 (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA6308Δ2) or three (CphA6308Δ3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA6308. In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA6308Δ1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26°C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA6308 and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA6308Δ1.

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris

1985 ◽  
Vol 5 (5) ◽  
pp. 1111-1121
Author(s):  
S B Ellis ◽  
P F Brust ◽  
P J Koutz ◽  
A F Waters ◽  
M M Harpold ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeasts ◽  
Sequence Analyses ◽  
Yeast Pichia Pastoris ◽  
Oxidation Of Methanol ◽  
Regulated Expression

The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts. The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined. The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-responsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation.

scholarly journals Jak1 Plays an Essential Role for Receptor Phosphorylation and Stat Activation in Response to Granulocyte Colony-Stimulating Factor

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 597-604 ◽  
Author(s):  
Kazuya Shimoda ◽  
Jian Feng ◽  
Hiroshi Murakami ◽  
Shigekazu Nagata ◽  
Diane Watling ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Mutant Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Receptor Phosphorylation ◽  
Signal Transducers ◽  
The Family ◽  
Proliferation And Differentiation ◽  
Stimulating Factor

Abstract The proliferation and differentiation of neutrophils is regulated by granulocyte-specific colony-stimulating factor (G-CSF ). G-CSF uses a receptor of the cytokine receptor superfamily and, in common with all members of the family, induces the tyrosine phosphorylation and activation of members of the Janus protein tyrosine kinase (Jak) family. In both myeloid cells and a human fibrosarcoma cell line expressing the G-CSF receptor, G-CSF induces the tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2. In addition, G-CSF induces the tyrosine phosphorylation of the receptor and members of the signal transducers and activators of transcription (Stat) family, including Stat3, as well as Stat1 and Stat5, depending on the cells involved. Using mutant cell lines lacking various Jaks, we show here that Jak1 is critical for G-CSF–mediated Stat activation, whereas Jak2 or Tyk2 are either not required or play redundant or ancillary roles. In the absence of Jak1, G-CSF induces activation of Jak2 and Tyk2, but fails to induce receptor tyrosine phosphorylation and induces dramatically reduced levels of Stat activation. A kinase-inactive Jak2, when overexpressed in cells lacking endogenous Jak2, can suppress Jak1 activation, receptor phosphorylation, and Stat activation, suggesting competition in the receptor complex either for Jak1 binding or substrates. Because the requirement for Jak1 is very similar to that previously shown for interleukin-6 signaling, the data support the concept that the G-CSF receptor and gp130 are both structurally and functionally similar.

scholarly journals FAT10 is a proteasomal degradation signal that is itself regulated by ubiquitination

2012 ◽  
Vol 23 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Samuel Buchsbaum ◽  
Beatrice Bercovich ◽  
Aaron Ciechanover
Keyword(s):  
Mutant Cell ◽  
Proteasomal Degradation ◽  
Posttranslational Regulation ◽  
Inflammatory Stimuli ◽  
Induction Of Apoptosis ◽  
In Cells

FAT10 is a ubiquitin-like protein modifier that is induced in vertebrates following certain inflammatory stimuli. Its functions and the repertoire of its target substrates have remained elusive. In contrast to ubiquitin, its cellular abundance is tightly controlled by both transcriptional and posttranslational regulation, and it was reported to be rapidly degraded by the proteasome. Here we provide data to indicate that the degradation of FAT10 requires ubiquitination: degradation was inhibited in cells expressing a ubiquitin mutant that cannot be polymerized and in a mutant cell harboring a thermolabile ubiquitin-activating enzyme, E1. Of importance, FAT10 can serve as a degradation signal for otherwise stable proteins, and in this case, too, the targeting to the proteasome requires ubiquitination. Degradation of FAT10 is accelerated after induction of apoptosis, suggesting that it plays a role in prosurvival pathways.

scholarly journals High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors

2020 ◽  
Vol 104 (21) ◽  
pp. 9205-9218
Author(s):  
Nina Jankowski ◽  
Katja Koschorreck ◽  
Vlada B. Urlacher
Keyword(s):  
Pichia Pastoris ◽  
Alcohol Oxidase ◽  
Building Blocks ◽  
Pleurotus Eryngii ◽  
Oxidation Products ◽  
Oxidative Enzymes ◽  
Biotechnological Applications ◽  
Positive Health ◽  
Neuroprotective Effects ◽  
Flavor Compound

Abstract The fungal secretome comprises various oxidative enzymes participating in the degradation of lignocellulosic biomass as a central step in carbon recycling. Among the secreted enzymes, aryl-alcohol oxidases (AAOs) are of interest for biotechnological applications including production of bio-based precursors for plastics, bioactive compounds, and flavors and fragrances. Aryl-alcohol oxidase 2 (PeAAO2) from the fungus Pleurotus eryngii was heterologously expressed and secreted at one of the highest yields reported so far of 315 mg/l using the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). The glycosylated PeAAO2 exhibited a high stability in a broad pH range between pH 3.0 and 9.0 and high thermal stability up to 55 °C. Substrate screening with 41 compounds revealed that PeAAO2 oxidized typical AAO substrates like p-anisyl alcohol, veratryl alcohol, and trans,trans-2,4-hexadienol with up to 8-fold higher activity than benzyl alcohol. Several compounds not yet reported as substrates for AAOs were oxidized by PeAAO2 as well. Among them, cumic alcohol and piperonyl alcohol were oxidized to cuminaldehyde and piperonal with high catalytic efficiencies of 84.1 and 600.2 mM−1 s−1, respectively. While the fragrance and flavor compound piperonal also serves as starting material for agrochemical and pharmaceutical building blocks, various positive health effects have been attributed to cuminaldehyde including anticancer, antidiabetic, and neuroprotective effects. PeAAO2 is thus a promising biocatalyst for biotechnological applications. Key points • Aryl-alcohol oxidase PeAAO2 from P. eryngii was produced in P. pastoris at 315 mg/l. • Purified enzyme exhibited stability over a broad pH and temperature range. • Oxidation products cuminaldehyde and piperonal are of biotechnological interest. Graphical abstract

Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris

2013 ◽  
Vol 166 (4) ◽  
pp. 174-181 ◽  
Author(s):  
Sehoon Kim ◽  
Shannon Warburton ◽  
Istvan Boldogh ◽  
Cecilia Svensson ◽  
Liza Pon ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Alcohol Oxidase ◽  
Peroxisome Biogenesis ◽  
Aox1 Promoter ◽  
Fermentation Processes

scholarly journals Kinase Screening in Pichia pastoris Identified Promising Targets Involved in Cell Growth and Alcohol Oxidase 1 Promoter (PAOX1) Regulation

PLoS ONE ◽  
2016 ◽  
Vol 11 (12) ◽  
pp. e0167766 ◽  
Author(s):  
Wei Shen ◽  
Chuixing Kong ◽  
Ying Xue ◽  
Yiqi Liu ◽  
Menghao Cai ◽  
...  
Keyword(s):  
Cell Growth ◽  
Pichia Pastoris ◽  
Alcohol Oxidase ◽  
Kinase Screening

scholarly journals Interaction of a Host Protein with Core Complexes of Bacteriophage Φ6 To Control Transcription

2010 ◽  
Vol 84 (9) ◽  
pp. 4821-4825 ◽  
Author(s):  
Xueying Qiao ◽  
Yang Sun ◽  
Jian Qiao ◽  
Leonard Mindich
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Polymerase ◽  
Fluorescent Protein ◽  
Host Protein ◽  
Genomic Segment ◽  
Double Stranded Rna ◽  
Infection Period ◽  
The Family ◽  
Green Fluorescent ◽  
In Cells

ABSTRACT Bacteriophages of the family Cystoviridae have genomes consisting of three double-stranded RNA (dsRNA) segments, L, S, and M, packaged within a polyhedral capsid along with RNA polymerase. Transcription of genomic segment L is activated by the interaction of host protein YajQ with the capsid structure. Segment L codes for the proteins of the inner capsid, which are expressed early in infection. Green fluorescent protein (GFP) fusions with YajQ produce uniform fluorescence in uninfected cells and in cells infected with viruses not dependent on YajQ. Punctate fluorescence develops when cells are infected with YajQ-dependent viruses. It appears that the host protein binds to the infecting particles and remains with them during the entire infection period.

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