Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.     

Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon.     

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.     

A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan

Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY∗ is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY∗, which carries a mutation homologous to yeast CPY∗, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY∗-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY∗-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY∗-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.     

Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein

Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx_40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM₁ ganglioside receptor of LTB. The rLTB.Etx_40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx_40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens.

Abbreviations

aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.     

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis^1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.     

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways^1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus egg extract. One method is visually informative ([³⁵S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.     

Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila

The Atg2–Atg18 complex acts in parallel to Atg8 and regulates Atg9 recycling from phagophore assembly site (PAS) during autophagy in yeast. Here we show that in Drosophila, both Atg9 and Atg18 are required for Atg8a puncta formation, unlike Atg2. Selective autophagic degradation of ubiquitinated proteins is mediated by Ref(2)P/p62. The transmembrane protein Atg9 accumulates on refractory to Sigma P (Ref(2)P) aggregates in Atg7, Atg8a and Atg2 mutants. No accumulation of Atg9 is seen on Ref(2)P in cells lacking Atg18 or Vps34 lipid kinase function, while the Atg1 complex subunit FIP200 is recruited. The simultaneous interaction of Atg18 with both Atg9 and Ref(2)P raises the possibility that Atg18 may facilitate selective degradation of ubiquitinated protein aggregates by autophagy.     

Comparative Analysis of the Conserved Functions of Arabidopsis DRL1 and Yeast KTI12

Patterning of the polar axis during the early leaf developmental stage is established by cell-to-cell communication between the shoot apical meristem (SAM) and the leaf primordia. In a previous study, we showed that the DRL1 gene, which encodes a homolog of the Elongator-associated protein KTI12 of yeast, acts as a positive regulator of adaxial leaf patterning and shoot meristem activity. To determine the evolutionally conserved functions of DRL1, we performed a comparison of the deduced amino acid sequence of DRL1 and its yeast homolog, KTI12, and found that while overall homology was low, well-conserved domains were presented. DRL1 contained two conserved plant-specific domains. Expression of the DRL1 gene in a yeast KTI12-deficient yeast mutant suppressed the growth retardation phenotype, but did not rescue the caffeine sensitivity, indicating that the role of Arabidopsis Elongator-associated protein is partially conserved with yeast KTI12, but may have changed between yeast and plants in response to caffeine during the course of evolution. In addition, elevated expression of DRL1 gene triggered zymocin sensitivity, while overexpression of KTI12 maintained zymocin resistance, indicating that the function of Arabidopsis DRL1 may not overlap with yeast KTI12 with regards to toxin sensitivity. In this study, expression analysis showed that class-I KNOX genes were downregulated in the shoot apex, and that YAB and KAN were upregulated in leaves of the Arabidopsis drl1-101 mutant. Our results provide insight into the communication network between the SAM and leaf primordia required for the establishment of leaf polarity by mediating histone acetylation or through other mechanisms.     

Differential physiological roles of ESCRT complexes in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Endosomal sorting complex required for transport (ESCRT) complexes are involved in endosomal trafficking to the lysosome, cytokinesis, and viral budding. Extensive genetic, biochemical, and structural studies on the ESCRT system have been carried out in yeast and mammalian systems. However, the question of how the ESCRT system functions at the whole organism level has not been fully explored. In C. elegans, we performed RNAi experiments to knock-down gene expression of components of the ESCRT system and profiled their effects on protein degradation and endocytosis of YP170, a yolk protein. Targeted RNAi knock-down of ESCRT-I (tsg-101 and vps-28) and ESCRT-III (vps-24, and vps-32.2) components interfered with protein degradation while knock-down of ESCRT-II (vps-25 and vps-36) and ESCRT-III (vps-20 and vps-24) components hampered endocytosis. In contrast, the knockdown of vps-37, another ESCRT-I component, showed no defect in either YP170 uptake or degradation. Depletion of at least one component from each complex — ESCRT-0 (hgrs-1), ESCRT-I (tsg-101, vps-28, and vps-37), ESCRT-II (vps-36), ESCRT-III (vps-24), and Vps4 (vps-4) — resulted in abnormal distribution of embryos in the uterus of worms, possibly due to abnormal ovulation, fertilization, and egglaying. These results suggest differential physiological roles of ESCRT-0, -I, -II, and -III complexes in the context of the whole organism, C. elegans.     

Expression of the HSP24 gene from Trichoderma harzianum in Saccharomyces cerevisiae

Hsp24 is a small heat-shock protein (sHSP). Such proteins are important endogenous cytoprotection factors involved in defense. A 1116-bp full-length cDNA of the Hsp24, with a 645-bp open reading frame nucleotide encoding a 24-kDa polypeptide consisting of 214 amino acid residues, was isolated from Trichoderma harzianum. Sequence analysis revealed that Hsp24 gene has more than 42–58% amino acid sequence homology with those of other fungi. The Hsp24 gene was integrated into pYES2 by inserting into a single site for recombination, yielding the recombinant of pYES2/Hsp24. Hsp24 expressed by pYES2/Hsp24 was induced by galactose. We tested whether Hsp24 could confer abiotic stress resistance when it was introduced into yeast cells. A transgenic yeast harboring T. harzianum Hsp24 was generated under the control of a constitutively expressed GAL promoter. The results indicated that Hsp24 yeast transformants had significantly higher resistance to salt, drought and heat stresses.     

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions

The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP^Sc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.     

Atg8 is essential for macropexophagy in Hansenula polymorpha

We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen-limitation induced microautophagy. Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole. After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome. In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm-like opening remained. We hypothesize that H. polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.     

Genomic Insights into the Saccharomyces sensu stricto Complex

The Saccharomyces sensu stricto group encompasses species ranging from the industrially ubiquitous yeast Saccharomyces cerevisiae to those that are confined to geographically limited environmental niches. The wealth of genomic data that are now available for the Saccharomyces genus is providing unprecedented insights into the genomic processes that can drive speciation and evolution, both in the natural environment and in response to human-driven selective forces during the historical “domestication” of these yeasts for baking, brewing, and winemaking.     

Amino acid composition of leaf,grain and bracts of japonica rice (<Emphasis Type="Italic">Oryza Sativa</Emphasis> ssp. <Emphasis Type="Italic">japonica</Emphasis>) and its response to nitrogen fertilization

Heat stress is one of the main abiotic stresses that limit plant growth. The effects of high temperature on oxidative damage, PSII activity and D1 protein turnover were studied in three wheat varieties with different heat susceptibility (CS, YN949 and AK58). The results showed that heat stress induced lower lipid peroxidation in AK58 and YN949 than CS, which was related to different changes of SOD, CAT, POD and H₂O₂. Similarly, AK58 and YN949 performed better PSII photochemical efficiency (F_v/F_m, ΦPSII and ETR) under high temperature, which was attributed to rapid synthesis and degradation of D1 protein. Moreover, higher expression of D1 protein turnover-related genes (PsbA, STN8, PBCP, Deg1, Deg2, Deg5, Deg8, FtsH1/5 and FtsH2/8) and SOD activity in AK58 and YN949 under normal conditions also established a basis for acclimatizing high temperatures, thereby alleviating PSII photoinhibition and reducing oxidative damage when exposed to heat stress.