Protein degradation and dynamic tRNA thiolation fine-tune translation at elevated temperatures.

Maintenance of protein quality control has implications in various processes such as neurodegeneration and ageing. To investigate how environmental insults affect this process, we analysed the proteome of yeast continuously exposed to mild heat stress. In agreement with previous transcriptomics studies, amongst the most marked changes, we found up-regulation of cytoprotective factors; a shift from oxidative phosphorylation to fermentation; and down-regulation of translation. Importantly, we also identified a novel, post-translationally controlled, component of the heat shock response. The abundance of Ncs2p and Ncs6p, two members of the URM1 pathway responsible for the thiolation of wobble uridines in cytoplasmic tRNAs tK(UUU), tQ(UUG) and tE(UUC), is down-regulated in a proteasomal dependent fashion. Using random forests we show that this results in differential translation of transcripts with a biased content for the corresponding codons. We propose that the role of this pathway in promoting catabolic and inhibiting anabolic processes, affords cells with additional time and resources needed to attain proper protein folding under periods of stress.

tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding.

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK(UUU)), (tQ(UUG)), and (tE(UUC)). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK(UUU) to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.

VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase.

BACKGROUND: FAF1 is a ubiquitin-binding adaptor for the p97 ATPase and belongs to the UBA-UBX family of p97 cofactors. p97 converts the energy derived from ATP hydrolysis into conformational changes of the p97 hexamer, which allows the dissociation of its targets from cellular structures or from larger protein complexes to facilitate their ubiquitin-dependent degradation. VAPB and the related protein VAPA form homo- and heterodimers that are anchored in the endoplasmic reticulum membrane and can interact with protein partners carrying a FFAT motif. Mutations in either VAPB or p97 can cause amyotrophic lateral sclerosis, a neurodegenerative disorder that affects upper and lower motor neurons. RESULTS: We show that FAF1 contains a non-canonical FFAT motif that allows it to interact directly with the MSP domain of VAPB and, thereby, to mediate VAPB interaction with p97. This finding establishes a link between two proteins that can cause amyotrophic lateral sclerosis when mutated, VAPB/ALS8 and p97/ALS14. Subsequently, we identified a similar FFAT-like motif in the ASNA1 subunit of the transmembrane-domain recognition complex (TRC), which in turn mediates ASNA1 interaction with the MSP domain of VAPB. Proteasome inhibition leads to the accumulation of ubiquitinated species in VAPB immunoprecipitates and this correlates with an increase in FAF1 and p97 binding. We found that VAPB interaction with ubiquitinated proteins is strongly reduced in cells treated with FAF1 siRNA. Our efforts to determine the identity of the ubiquitinated targets common to VAPB and FAF1 led to the identification of RPN2, a subunit of an oligosaccharyl-transferase located at the endoplasmic reticulum, which may be regulated by ubiquitin-mediated degradation. CONCLUSIONS: The FFAT-like motifs we identified in FAF1 and ASNA1 demonstrate that sequences containing a single phenylalanine residue with the consensus (D/E)(D/E)FEDAx(D/E) are also proficient to mediate interaction with VAPB. Our findings indicate that the repertoire of VAPB interactors is more diverse than previously anticipated and link VAPB to the function of ATPase complexes such as p97/FAF1 and ASNA1/TRC.

Hydrophilic strong anion exchange (hSAX) chromatography for highly orthogonal peptide separation of complex proteomes.

Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.

Pseudoxanthomonas indica sp. nov., isolated from a hexachlorocyclohexane dumpsite.

A bacterial strain, designated P15(T), was isolated from the soil of an open hexachlorocyclohexane dumpsite. Comparative sequence analysis showed that strain P15(T) displayed high 16S rRNA gene sequence similarities (94.4-97.2 %) with members of the genus Pseudoxanthomonas. The isolate was most closely related to Pseudoxanthomonas mexicana AMX 26B(T) (97.2 % 16S rRNA gene sequence similarity) and Pseudoxanthomonas japonensis 12-3(T) (97.2 %). DNA-DNA relatedness studies showed unambiguously that strain P15(T) represented a novel species that was separate from P. mexicana DSM 17121(T) (7.7 %) and P. japonensis DSM 17109(T) (9.4 %). The predominant cellular fatty acids of strain P15(T) were iso-C16:0 (21.4 %), iso-C15:0 (16.1 %), summed feature 9 (comprising iso-C17:1ω9c and/or 10-methyl C16:0; 14.9 %), iso-C11:0 3-OH (8.3 %) and iso-C14:0 (7.0 %). The polar lipid profile of strain P15(T) showed the presence of large amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol in addition to unknown glycolipids, phospholipids and an amino-group-containing polar lipid. Ubiquinone 8 was found as the major quinone. The polyamine profile showed the presence of spermidine. The DNA G+C content was 62.9±2 mol%. Strain P15(T) is described as representing a new member of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas indica sp. nov. is proposed. The type strain is P15(T) ( = MTCC 8596(T) = CCM 7430(T)).