Inhibiting K63 Polyubiquitination Abolishes No-Go Type Stalled Translation Surveillance in <i>Saccharomyces cerevisiae</i>

<div><p>Incidental ribosome stalling during translation elongation is an aberrant phenomenon during protein synthesis and is subjected to quality control by surveillance systems, in which mRNA and a nascent protein are rapidly degraded. Their detailed molecular mechanisms as well as responsible factors for these processes are beginning to be understood. However, the initial processes for detecting stalled translation that result in degradation remain to be determined. Among the factors identified to date, two E3 ubiquitin ligases have been reported to function in distinct manners. Because ubiquitination is one of the most versatile of cellular signals, these distinct functions of E3 ligases suggested diverse ubiquitination pathways during surveillance for stalled translation. In this study, we report experimental evidences for a unique role of non-proteasomal K63 polyubiquitination during quality control for stalled translation. Inhibiting K63 polyubiquitination by expressing a K63R ubiquitin mutation in <i>Saccharomyces cerevisiae</i> cells markedly abolished the quality control responses for stalled translation. More detailed analyses indicated that the effects of K63R mutants were independent of the proteasome and that K63 polyubiquitination is dependent on Hel2, one of the E3 ligases. Moreover, a K63R ubiquitin mutant barely inhibited the quality control pathway for nonstop translation, indicating distinct mechanisms for these highly related quality control pathways. Our results suggest that non-proteasomal K63 polyubiquitination is included in the initial surveillance process of stalled translation and presumably triggers protein degradation steps upon translational stall. These findings provide crucial information regarding the detailed molecular mechanisms for the initial steps involved in quality control systems and their classification.</p></div

Effects of proteasome inhibitors, MG132 and PS341, on stalled translation and K63R expression.

<p>(A) Western blot analysis for effect of MG132. Vectors with Rluc-HIS3 reporters were introduced into a wild-type strain (BY4727). Yeast transformants were incubated for 5 h with either DMSO (e.g., 0 μM MG132) or 75 μM MG132. Arrowhead 1 indicates the Rluc protein only and arrowhead 2 indicates the Rluc-blank-HIS3 protein. PGK1 was used as a loading control. (B) Dual luciferase assay for the effect of MG132 on wild-type strain with Rluc-CGA x12-luc2 reporter (HRKW-2). MG132 was used as in (A). (C) The empty or K63R ubiquitin mutation plasmids were introduced into the <i>pdr5</i><b><i>∆</i></b> yeast strain harboring Rluc-luc2 reporter (SKY142). The transformants were incubated in the presence of MG132 or PS341 at indicated concentrations for 2 h. Fold differences between the percentages of luc2/Rluc ratios between the transformants with the absence (-K63R) or presence (+K63R) of K63R expression are indicated over the top of the bar graphs.</p

Effect of K63 polyubiquitination on the quality control for stalled translation.

<p>(A) Structure of Ubiquitin protein and location of the lysine residues for polyubiquitination [id: 1UBQ]. K63 (red) and other lysine residues (pink) are shown in sticks. The structural model was generated using MolFeat version 3.5 (Fiatlux, Tokyo, Japan). (B) Schematic illustration for the inhibition of polyubiquitination by arginine mutants. Arginine mutants were expressed from expression plasmids in yeast cells harboring endogenous ubiquitin genes on its genome. An incorporation of an arginine mutant into the (poly-) ubiquitin chain terminates further elongation. (C) Cellular ubiquitination levels of wild-type and ubiquitin mutants with a single arginine substitution (K6R, K11R, K27R, K29R, K33R, K48R, K63R). Ubiquitin genes were fused with His-tag and expressed from plasmids in the wild-type strain (BY4727). Cellular ubiquitination levels were detected by antibody for His-tag. PGK antibody was used as loading control. (D) Dual luciferase assay for the effects of arginine mutants of ubiquitin protein on the Rluc-CGA x12-luc2 reporter. Plasmids that included ubiquitin arginine mutants were introduced into a wild-type strain (HRKW-2). Ubi-WT indicates wild-type ubiquitin. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements.</p

Isotopic Combinatomer Analysis Provides <i>in Vivo</i> Evidence of the Direct Epimerization of Monoglucosyl Diacylglycerol in Cyanobacteria

Galactolipids constitute the majority of photosynthetic membranes called thylakoid membranes in cyanobacteria and chloroplasts of land plants and algae. The galactolipids, although identical in headgroup structure, are synthesized by significantly different pathways in cyanobacteria and chloroplasts. In the cyanobacterial pathway, monoglucosyl diacylglycerol (GlcDG) is synthesized first and then converted to monogalactosyl diacylglycerol (MGDG). On the basis of circumstantial evidence, the mechanism of conversion was thought to be epimerization at C-4, but no direct evidence has yet been provided, because there is no <i>in vitro</i> enzymatic system of the putative membrane-bound reaction. Labeling studies with ¹⁴C and ¹³C suggested that the labels in the headgroup and the acyl groups were kept at a reasonably constant ratio before and after the conversion. We then provide <i>in vivo</i> evidence of the direct epimerization based on detailed isotopomer analysis of the conversion, named “combinatomer analysis”. The different types of molecules formed by the combination of labeled or unlabeled parts (<i>sn</i>-1 acyl, <i>sn</i>-2 acyl, glycerol, and hexose) are called here “combinatomers”. Combinatomer analysis of the experiments with pulse labeling with ¹³C and chase in <i>Anabaena</i> sp. PCC 7118 indicated that the composition of combinatomers in the precursor GlcDG was kept unchanged in the product MGDG. Production of combinatomers resulting from exchange of hexose was minimal. This provides solid evidence of the epimerization of the glucose moiety of GlcDG, as well as the direct desaturation of acyl groups at the <i>sn</i>-1 position

Comparisons of the effects of deleting Hel2 and Ltn1.

<p>(A) Western blot analysis for Rluc-HIS3 reporters from the wild-type (WT), <i>hel2</i><b><i>∆</i></b>, and <i>ltn1</i><b><i>∆</i></b> strains (BY4727, SKY61, S18-E01). A schematic image of reporter fusion genes, Rluc-HIS3, either with or without 12 CGA codon repeats, is illustrated above. The expression of fusion protein was detected using a Rluc antibody. PGK1 was used as a loading control. Arrowhead 1 indicates the Rluc protein alone, arrowhead 2 indicates the Rluc-blank-HIS3 protein, and arrowhead 3 indicates the Rluc-CGA x12-HIS3 protein. (B) Dual luciferase assay for Rluc-CGA x12-luc2 reporter in the wild-type (WT), <i>hel2</i><b><i>∆</i></b>, and <i>ltn1</i><b><i>∆</i></b> strains (HRKW-2, SKY113, HRKW-6). A schematic image of reporter fusion genes, Rluc-luc2, either with or without 12 CGA codon repeats, is illustrated above. Bars indicate luc2/Rluc ratios. Percentages were standardized using the results of a dual luciferase assay for Rluc-blank-luc2 in the wild-type strain (HRKW-1). The inset indicates the results with the <i>ltn1</i><b><i>∆</i></b> strain with an appropriately adjusted range. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements.</p

Effects of K0 and K63only ubiquitin mutants on stalled translation.

<p>(A) Cellular ubiquitination levels of K0 and K63only ubiquitin mutants. Ubiquitinated proteins were detected as above. Amount of the protein sample from a cell expressing His-tagged wild-type ubiquitin are decreased to 1/16 and 1/4 dilution. (B) Dual luciferase assay for the of K0 and K63only ubiquitin mutants on the Rluc-luc2 reporter. Plasmids bearing K0 or K63only ubiquitin mutants were introduced into a wild-type strain harboring Rluc-luc2 reporter strain (HRKW-2). (single) indicates the transformant with ubiquitin expression from single copy plasmids, and (multi) indicates that from multiple copy plasmids. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements. (C) Genetic colony growth test for the effect of K0 and K63only ubiquitin mutants on the Rluc-HIS3 reporter (SKY24). Types of ubiquitin vectors are indicated on the left. Transformant colonies were streaked on the SC-Leucine (Leu) (middle), and SC-Leucine, Histidine (Leu, His) (right) plates, and colony growth was monitored for 4 days at 30°C.</p

Temporal regulation of K63 polyubiquitination and Hel2 with other factors for stalled translation.

<p>(A) The luc2/Rluc ratios for Rluc-luc2 reporter in the wild-type (WT) and single knockout strains (as indicated) in the presence (+K63R) or absence (-K63R) of K63R expression. The inset shows the results for the <i>rqc1</i><b><i>∆</i></b> and <i>ltn1</i><b><i>∆</i></b> strains. The average luc2/Rluc ratios and their standard deviations were obtained for three independent measurements. (B) The luc2/Rluc ratios for Rluc-luc2 reporter in the strains with double knockout of Hel2 and other factors involved in stalled translation. All strains are <i>hel2</i><b><i>∆</i></b>. Thus, as examples, WT in this figure indicates a <i>hel2</i><b><i>∆</i></b> single knockout strain, and <i>asc1</i><b><i>∆</i></b> indicates the <i>hel2</i><b><i>∆</i></b><i>asc1</i><b><i>∆</i></b> strain. The average luc2/Rluc ratios and their standard deviations were obtained for three independent measurements. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

Top-down Metabolomic Approaches for Nitrogen-Containing Metabolites

Streamlining the processes that reveal heteroatom-containing metabolites and their biosynthetic genes is essential in integrated metabolomics studies. These metabolites are especially targeted for their potential pharmaceutical activities. By using a Fourier-transform ion cyclotron resonance–mass spectrometry (FTICR–MS) instrument, we provide top-down targeted metabolomic analyses using ultrahigh-resolution liquid chromatography–mass spectrometry (LC–MS), high-resolution matrix-assisted laser desorption/ionization (MALDI), and high-resolution imaging mass spectrometry (IMS) with ¹⁵N labeling of nitrogen-containing metabolites. In this study, we efficiently extract known and unknown chemicals and spatial information from the medicinal plant Catharanthus roseus, which sources several cancer drugs. The ultrahigh-resolution LC–MS analysis showed that the molecular formula of 65 N-metabolites were identified using the petals, peduncles, leaves, petioles, stems, and roots of the non- and ¹⁵N-labeled Catharanthus plants. The high resolution MALDI analysis showed the molecular formula of 64 N-metabolites using the petals, leaves, and stems of the non- and ¹⁵N-labeled Catharanthus. The chemical assignments using molecular formulas stored in databases identified known and unknown metabolites. The comparative analyses using the assigned metabolites revealed that most of the organ-specific ions are derived from unknown N-metabolites. The high-resolution IMS analysis characterized the spatial accumulation patterns of 32 N-metabolites using the buds, leaves, stems, and roots in Catharanthus. The comparative analysis using the non- and ¹⁵N-labeled IMS data showed the same spatial accumulation patterns of a non- and ¹⁵N-labeled metabolite in the organs, showing that top-down analysis can be performed even in IMS analysis

Calculation results for the late stage of the biosynthesis of anthocyanidin in a neutral aqueous environment.

<p>Calculation results for the late stage of the biosynthesis of anthocyanidin in a neutral aqueous environment.</p

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