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Little is known about the impact of smoking on voice acoustics. Some studies have found that tobacco affects the fundamental frequency of the voice, whereas others have not. This study aimed to overcome the major methodological limitations observed in previous studies by strictly controlling several variables that could clarify the effect of smoking on the speaking voice. Lebanese men were chosen for this study. Thirty nonsmokers, 30 cigarette smokers, and 30 water-pipe smokers were matched on the basis of age, height, and weight. The 90 participants were asked to complete the Voice Handicap Index, sustain the vowel /a/, read 10 sentences in French and Arabic, and speak spontaneously in both languages. The mean fundamental frequency (F0), speaking fundamental frequency (SFF), jitter, and standard deviation of F0 were measured using Praat and Vocalab4 and were compared between the groups. The Voice Handicap Index scores differed significantly between nonsmokers and cigarette smokers and between nonsmokers and water-pipe smokers. Results also show that cigarette smokers' F0 and SFF were significantly lower than nonsmokers' results. No significant differences were found between water-pipe smokers and nonsmokers. The jitter and the standard deviation of F0 did not differ significantly between the two groups. Our findings clearly demonstrate the effect of smoking on the voice: smokers reported more voice complaints, and cigarette smokers presented lower F0 and SFF in French and in Arabic when age, height, and weight were controlled. Further investigations using similar strict controls over individual variables and additional measures are encouraged to better understand the effect of water-pipe smoking on the voice. [ABSTRACT FROM AUTHOR]

Transgender MtF people (trans women) consult otorhinolaryngologists and vocologists with the aim of feminizing their voice and being consistently perceived as women. Treatment of these trans women always begins with a vocal assessment that is relatively unspecific as it was originally constructed for individuals with dysphonia. This study examines the subjective portion of the assessment and specifically the self-assessment questionnaire. There is no French-language questionnaire designed to identify the issues facing people who want voice feminization and quantify the impact of their voice disorder on their daily lives. We present a translation and adaption into French of the questionnaire developed by Dacakis et al [6]. This work follows the World Health Organization recommendations [12] regarding translation. Thirty-six Belgian and French trans women took part in this study. The results show excellent repeatability and reliability, while the construct validity measures show that the items correlate with six areas of concern for trans women identified by Davies and Johnson [7] in a previous study. The domains are the following: effect of voice on ease of social interaction, effect of voice on emotions, relationship between voice and gender identity, effort and concentration required to produce voice, physical aspects of voice production, and pitch. Concurrent validity could not be measured owing to lack of sufficiently detailed stories. The psychometric properties of the French version of the questionnaire are acceptable. The questionnaire can be used as is in daily clinical practice. [ABSTRACT FROM AUTHOR]

Summary Background The original Dysphonia Severity Index (ie, DSI) weighs and combines four voice markers in a single number to size dysphonia gradation in the clinic: percent jitter (from Multi-Dimensional Voice Program; KayPENTAX Corp., Montvale, NJ), softest intensity and highest fundamental frequency (both from Voice Range Profile; KayPENTAX Corp.), and maximum phonation time. To be more generally applied, however, implementation of DSI in the program Praat (Paul Boersma and David Weenink, Institute for Phonetic Sciences, University of Amsterdam, The Netherlands) would be advantageous for all voice clinicians. The presented project was therefore designed (1) to develop such a Praat application and (b) to test its concurrent validity. Methods The four voice markers for the original DSI, as well as ten additional voice markers in Praat , were administered on a total of 49 subjects in three different clinical voice centers. A crossover research design was implemented to counterbalance for possible exercise effects. First, stepwise multiple linear regression was applied to build a statistical model with the best combination of Praat predictors for the original DSI. Second, correlation statistics were applied to compare Praat 's DSI with the original DSI. Results Both DSI versions correlated strongly. A custom script was therefore written for automated DSI determination in Praat . Conclusion With this script, every voice clinician can easily determine DSI in the Praat program. [ABSTRACT FROM AUTHOR]

Summary Objective The purposes of this study were to assess the long-term efficacy of medialization laryngoplasty via injection of autologous fat in patients with unilateral laryngeal paralysis, and to discuss the results based on the volume of fat injected and the size of the initial glottic gap. Study design This was a retrospective study with clinical reevaluation of 18 patients treated for unilateral laryngeal paralysis at Liège University Hospital between April 1, 2011, and December 1, 2014. Method The voice examination included subjective scales (GRBAS and the Voice Handicap Index), a report on acoustic and aerodynamic voice parameters, and laryngostroboscopic assessment of glottic closure and mucosal wave (T0: preoperative; T1: immediate postoperative; T2: >1 year). Results The median follow-up duration was 19 months. We observed a significant improvement in maximum phonation time; mean flow rate; jitter; the Voice Handicap Index; the G, R, B, and A subscales of the GRBAS-I; and glottic closure at T1. These parameters remained stable over time, without any significant difference except for shimmer and glottic closure between T1 and T2. Regardless of the volume of fat injected and the magnitude of the initial glottic gap, we did not see any significant difference from one assessment time to another. Conclusion Medialization laryngoplasty by means of the injection of autologous fat is a safe, effective surgical technique for patients with unilateral laryngeal paralysis regardless of the size of the initial glottic gap. The results are maintained longer than 1 year, and no evidence of significant resorption of the fat was found in our study over a period of 12–58 months. The volume of fat injected does not seem to affect the functional results. [ABSTRACT FROM AUTHOR]

Summary: Objective: Vocal accuracy of a sung performance can be evaluated by two methods: acoustic analyses and subjective judgments. Acoustic analyses have been presented as a more reliable solution but both methods are still used for the evaluation of singing voice accuracy. This article presents a first time direct comparison of these methods. Methods: One hundred sixty-six untrained singers were asked to sing the popular song “Happy Birthday.” These recordings constituted the database analyzed. Acoustic analyses were performed to quantify the pitch interval deviation, number of contour errors, and number of tonality modulations for each recording. Additionally, 18 experts in singing voice or music rated the global pitch accuracy of these performances. Results: A high correlation occurred between acoustic measurements and subjective rating. The total model of acoustic analyses explained 81% of the variance of the judges'' scores. Their rating was influenced by both tonality modulations and pitch interval deviation. Conclusions: This study highlights the congruence between objective and subjective measurements of vocal accuracy within this first time comparison. Our results confirm the relevance of the pitch interval deviation criterion in vocal accuracy assessment. Furthermore, the number of tonality modulations is also a salient criterion in perceptive rating and should be taken into account in studies using acoustic analyses. [Copyright &y& Elsevier]

Summary: Objectives: This study evaluates the effect of a 2-hour reading task between 70 and 75dB(A) in 16 normophonic and 16 dysphonic female teachers with vocal nodules. Methods: Objective measurements (acoustic analysis, voice range measurements, and aerodynamic measurements) and subjective self-ratings were collected before and every 30 minutes during the reading to determine the voice evolution in both groups. Results: Fundamental frequency, lowest frequency, highest frequency (F-High), highest intensity, and intensity range increase through the reading, whereas shimmer decreases. Maximum phonation time decreases after 30 minutes. Estimated subglottal pressure (ESP) and sound pressure level increase during the first hour. Afterward, ESP decreases. Self-ratings worsen through time. When comparing the normophonic and the dysphonic teachers, self-ratings reveal more complaints in the dysphonic group. Few differences in objective measurements are found between both groups: normophonic teachers show lower ESP, higher F-High, and greater frequency range. Conclusions: Frequency modifications from acoustic analysis and voice range measurements suggest an increased laryngeal tension during vocal load, while subjects perceive a worsening of voice. Aerodynamic parameters depict first a deterioration of voice efficiency and then an adaptation to the prolonged reading. The comparison between both groups shows a discrepancy between objective measurements and self-ratings, suggesting that both approaches are necessary to have a complete view of vocal load effects. Surprisingly, both groups behave similarly through vocal load, without more or quicker deterioration of voice in the dysphonic group. [Copyright &y& Elsevier]

Summary: Objectives: The objectives of this study are to evaluate the impact on voice of both duration and intensity level of 2 hours of continuous oral reading. Voice modifications accompanying changes in intensity level during prolonged reading tasks are analyzed. Methods: Fifty normophonic women undergo two sessions of voice loading in which the required intensity level of voice varied between 60–65dB(A) for the first session and 70–75dB(A) for the second session. The effects of loading on objective data (average fundamental frequency [F0], jitter%, shimmer%, noise-to-harmonic ratio, maximum phonation time, lowest frequency [F-Low], highest frequency [F-High], frequency range [Range], lowest intensity [I-Low] level, and highest intensity level) and self-ratings (voice quality, phonation effort, vocal fatigue, and laryngeal discomfort) are assessed every 30 minutes during the loading tasks. Results: Results indicate that average F0, F-Low, I-Low, maximum phonation time, feeling of phonation effort, vocal fatigue, and laryngeal discomfort increase during prolonged reading, whereas shimmer% and self-rating of voice quality decrease. Average F0, F-High, and Range are the only parameters influenced by the required intensity of vocal load; they are significantly higher in the 70- to 75-dB session compared with the 60- to 65-dB session. Concerning the subjective self-ratings, similar results for the four ratings used suggest that only one would suffice in future studies. Conclusions: These results confirm the importance of both duration and intensity level as loading factors, even if intensity level affects fewer variables than duration. [Copyright &y& Elsevier]

Summary: The aim of our study was to validate a Pediatric Voice Symptom Questionnaire (PVSQ) presenting with a parallel form for children and their parents. The items of the questionnaire were elaborated from the results of structured interviews with dysphonic children (DP) and normophonic children (NP) and their mothers and were tested for feasibility in a pilot study involving 42 normophonic children aged 5–13 years. The items were then administered in a test-retest mode to 333 children and their parents (154 boys and 179 girls with a mean age of 9 years, standard deviation: 1.8); 45 consulting DP, 34 nonconsulting dysphonics (NcDP), 163 NP, and 91 others. Classical statistical analyses and an item response modeling approach were used to analyze the results. High internal consistency and good test-retest stability were found. Significant differences between total score of the NP, DP, and NcDP groups were observed both in the children and the parents and also between parental and child score for the NP and NcDP groups (P <0.001–P =0.014). Correlations between child and parental scores were found only in the DP groups (r =0.478; P <0.001). Based on our results, the PVSQ is a valid and reliable instrument for the autoevaluation of dysphonia in the child population. [Copyright &y& Elsevier]

In their environment, cells must cope with mechanical stresses constantly. Among these, nanoscale deformations of plasma membrane induced by substrate nanotopography are now largely accepted as a biophysical stimulus influencing cell behavior and function. However, the mechanotransduction cascades involved and their precise molecular effects on cellular physiology are still poorly understood. Here, using homemade fluorescent nanostructured cell culture surfaces, we explored the role of Bin/Amphiphysin/Rvs (BAR) domain proteins as mechanosensors of plasma membrane geometry. Our data reveal that distinct subsets of BAR proteins bind to plasma membrane deformations in a membrane curvature radius-dependent manner. Furthermore, we show that membrane curvature promotes the formation of dynamic actin structures mediated by the Rho GTPase CDC42, the F-BAR protein CIP4, and the presence of PI(4,5)P 2 . In addition, these actin-enriched nanodomains can serve as platforms to regulate receptor signaling as they appear to contain interferon-γ receptor (IFNγ-R) and to lead to the partial inhibition of IFNγ-induced JAK/STAT signaling.

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photosynthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca 2+ or Mn 2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H + ions are transported in exchange for Ca 2+ and Mn 2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca 2+ , Mn 2+ or H + gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H + ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H + transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM., (© 2023 The Authors. Traffic published by John Wiley & Sons Ltd.)

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3, encoded by SH3GL3), which controls the cell surface homeostasis of the tumor marker CD166 (also known as ALCAM). Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here, we investigate the role of actin, Rho GTPases and microtubules, which are major players in CIE processes, in this mechanism. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers, and that its perturbation strongly inhibits the process of CD166 uptake. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci., Competing Interests: The authors of this manuscript have the following competing interests: PH is a Research Director at FNRS.

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

The coupling between mitochondrial respiration and photosynthesis plays an important role in the energetic physiology of green plants and some secondary-red photosynthetic eukaryotes (diatoms), allowing an efficient CO 2 assimilation and optimal growth. Using the flagellate Euglena gracilis, we first tested if photosynthesis-respiration coupling occurs in this species harbouring secondary green plastids (i.e. originated from an endosymbiosis between a green alga and a phagotrophic euglenozoan). Second, we tested how the trophic state (mixotrophy and photoautotrophy) of the cell alters the mechanisms involved in the photosynthesis-respiration coupling. Energetic coupling between photosynthesis and respiration was determined by testing the effect of respiratory inhibitors on photosynthesis, and measuring the simultaneous variation of photosynthesis and respiration rates as a function of temperature (i.e. thermal response curves). The mechanism involved in the photosynthesis-respiration coupling was assessed by combining proteomics, biophysical and cytological analyses. Our work shows that there is photosynthesis-respiration coupling and membrane contacts between mitochondria and chloroplasts in E. gracilis. However, whereas in mixotrophy adjustment of the chloroplast ATP/NADPH ratio drives the interaction, in photoautotrophy the coupling is conditioned by CO 2 limitation and photorespiration. This indicates that maintenance of photosynthesis-respiration coupling, through plastic metabolic responses, is key to E. gracilis functioning under changing environmental conditions., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Plasma membrane (PM) intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and small solutes. The functional importance of the PM organisation of PIPs in the interaction with other cellular structures is not completely understood. We performed a pull-down assay using maize (Zea mays) suspension cells expressing YFP-ZmPIP2;5 and validated the protein interactions by yeast split-ubiquitin and bimolecular fluorescence complementation assays. We expressed interacting proteins tagged with fluorescent proteins in Nicotiana benthamiana leaves and performed water transport assays in oocytes. Finally, a phylogenetic analysis was conducted. The PM-located ZmPIP2;5 physically interacts with the endoplasmic reticulum (ER) resident ZmVAP27-1. This interaction requires the ZmVAP27-1 cytoplasmic major sperm domain. ZmPIP2;5 and ZmVAP27-1 localise in close vicinity in ER-PM contact sites (EPCSs) and endocytic structures upon exposure to salt stress conditions. This interaction enhances PM water permeability in oocytes. Similarly, the Arabidopsis ZmVAP27-1 paralogue, AtVAP27-1, interacts with the AtPIP2;7 aquaporin. Together, these data indicate that the PIP2-VAP27 interaction in EPCSs is evolutionarily conserved, and suggest that VAP27 might stabilise the aquaporins and guide their endocytosis in response to salt stress., (©2020 The Authors. New Phytologist ©2020 New Phytologist Trust.)

The yeast Saccharomyces cerevisiae has been perceived over decades as a highly valuable model organism for the investigation of ion homeostasis. Indeed, many of the genes and biological systems that function in yeast ion homeostasis are conserved throughout unicellular eukaryotes to humans. In this context, measurement of the yeast cellular ionic content provides information regarding yeast response to gene deletion or exposure to chemicals for instance. We propose here a protocol that we tested for the analysis of 12 elements (Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cu 2+ , Fe 2+ , K + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Zn 2+ ) in yeast using Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). This technique enables determination of the cellular content of numerous ions from one biological sample., Competing Interests: Competing interestsThe authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

The Uncharacterized Protein Family 0016 (UPF0016) gathers poorly studied membrane proteins well conserved through evolution that possess one or two copies of the consensus motif Glu-x-Gly-Asp-(Arg/Lys)-(Ser/Thr). Members are found in many eukaryotes, bacteria and archaea. The interest for this protein family arose in 2012 when its human member TMEM165 was linked to the occurrence of Congenital Disorders of Glycosylation (CDGs) when harbouring specific mutations. Study of the UPF0016 family is undergone through the characterization of the bacterium Vibrio cholerae (MneA), cyanobacterium Synechocystis (SynPAM71), yeast Saccharomyces cerevisiae (Gdt1p), plant Arabidopsis thaliana (PAM71 and CMT1), and human (TMEM165) members. These proteins have all been identified as transporters of cations, more precisely of Mn 2+ , with an extra reported function in Ca 2+ and/or H + transport for some of them. Apart from glycosylation in humans, the UPF0016 members are required for lactation in humans, photosynthesis in plants and cyanobacteria, Ca 2+ signaling in yeast, and Mn 2+ homeostasis in the five aforementioned species. The requirement of the UPF0016 members for key physiological processes most likely derives from their transport activity at the Golgi membrane in human and yeast, the chloroplasts membranes in plants, the thylakoid and plasma membranes in cyanobacteria, and the cell membrane in bacteria. In the light of these studies on various UPF0016 members, this family is not considered as uncharacterized anymore and has been renamed the Gdt1 family according to the name of its S. cerevisiae member. This review aims at assembling and confronting the current knowledge in order to identify shared and distinct features in terms of transported molecules, mode of action, structure, etc., as well as to better understand their corresponding physiological roles., Competing Interests: Conflict of interest: The authors declare no conflicts of interest., (Copyright: © 2020 Thines et al.)

Cases of congenital disorders of glycosylation (CDG) have been associated with specific mutations within the gene encoding the human Golgi TMEM165 (transmembrane protein 165), belonging to UPF0016 (uncharacterized protein family 0016), a family of secondary ion transporters. To date, members of this family have been reported to be involved in calcium, manganese, and pH homeostases. Although it has been suggested that TMEM165 has cation transport activity, direct evidence for its Ca 2+ - and Mn 2+ -transporting activities is still lacking. Here, we functionally characterized human TMEM165 by heterologously expressing it in budding yeast ( Saccharomyces cerevisiae ) and in the bacterium Lactococcus lactis Protein production in these two microbial hosts was enhanced by codon optimization and truncation of the putatively autoregulatory N terminus of TMEM165. We show that TMEM165 expression in a yeast strain devoid of Golgi Ca 2+ and Mn 2+ transporters abrogates Ca 2+ - and Mn 2+ -induced growth defects, excessive Mn 2+ accumulation in the cell, and glycosylation defects. Using bacterial cells loaded with the fluorescent Fura-2 probe, we further obtained direct biochemical evidence that TMEM165 mediates Ca 2+ and Mn 2+ influxes. We also used the yeast and bacterial systems to evaluate the impact of four disease-causing missense mutations identified in individuals with TMEM165-associated CDG. We found that a mutation leading to a E108G substitution within the conserved UPF0016 family motif significantly reduces TMEM165 activity. These results indicate that TMEM165 can transport Ca 2+ and Mn 2+ , which are both required for proper protein glycosylation in cells. Our work also provides tools to better understand the pathogenicity of CDG-associated TMEM165 mutations., (© 2020 Stribny et al.)

Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O 2 - ) into molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn 2+ as well as to evaluate the redox state of the cell., Competing Interests: Competing interestsThe authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P)., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase., Competing Interests: The authors have declared that no competing interests exist.

The biological importance of manganese lies in its function as a key cofactor for numerous metalloenzymes and as non-enzymatic antioxidant. Due to these two essential roles, it appears evident that disturbed manganese homeostasis may trigger the development of pathologies in humans. In this context, yeast has been extensively used over the last decades to gain insight into how cells regulate intra-organellar manganese concentrations and how human pathologies may be related to disturbed cellular manganese homeostasis. This review first summarizes how manganese homeostasis is controlled in yeast cells and how this knowledge can be extrapolated to human cells. Several manganese-related pathologies whose molecular mechanisms have been studied in yeast are then presented in the light of the function of this cation as a non-enzymatic antioxidant or as a key cofactor of metalloenzymes. In this line, we first describe the Transmembrane protein 165-Congenital Disorder of Glycosylation (TMEM165-CDG) and Friedreich ataxia pathologies. Then, due to the established connection between manganese cations and neurodegeneration, the Kufor-Rakeb syndrome and prion-related diseases are finally presented., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The plasma membrane H + -ATPase of fungi and plants is a single polypeptide of fewer than 1,000 residues that extrudes protons from the cell against a large electric and concentration gradient. The minimalist structure of this nanomachine is in stark contrast to that of the large multi-subunit F O F 1 ATPase of mitochondria, which is also a proton pump, but under physiological conditions runs in the reverse direction to act as an ATP synthase. The plasma membrane H + -ATPase is a P-type ATPase, defined by having an obligatory phosphorylated reaction cycle intermediate, like cation pumps of animal membranes, and thus, this pump has a completely different mechanism to that of F O F 1 ATPases, which operates by rotary catalysis. The work that led to these insights in plasma membrane H + -ATPases of fungi and plants has a long history, which is briefly summarized in this review., (© 2018 The Authors. Yeast published by John Wiley & Sons, Ltd.)

The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO 2 fixation. Changes in free Ca 2+ in the stroma of chloroplasts have been observed at the transition between light and darkness, and also in response to stress stimuli. Such Ca 2+ dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca 2+ fluxes in the chloroplasts have been unknown. By employing a Ca 2+ reporter-based approach, we identified two chloroplast-localized Ca 2+ transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca 2+ signal in the chloroplast stroma. BICAT2 mediated Ca 2+ uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced [Ca 2+ ] stroma signal. Conversely, this Ca 2+ transient was increased in knockout mutants of BICAT1, which transports Ca 2+ into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected. These results show that BICAT transporters play a role in chloroplast Ca 2+ homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca 2+ transporters., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

By manipulating the various physicochemical properties of amino acids, the design of peptides with specific self-assembling properties has been emerging for more than a decade. In this context, short peptides possessing detergent properties (so-called "peptergents") have been developed to self-assemble into well-ordered nanostructures that can stabilize membrane proteins for crystallization. In this study, the peptide with "peptergency" properties, called ADA8 and extensively described by Tao et al., is studied by molecular dynamic simulations for its self-assembling properties in different conditions. In water, it spontaneously forms beta sheets with a β barrel-like structure. We next simulated the interaction of this peptide with a membrane protein, the bacteriorhodopsin, in the presence or absence of a micelle of dodecylphosphocholine. According to the literature, the peptergent ADA8 is thought to generate a belt of β structures around the hydrophobic helical domain that could help stabilize purified membrane proteins. Molecular dynamic simulations are here used to image this mechanism and provide further molecular details for the replacement of detergent molecules around the protein. In addition, we generalized this behavior by designing an amphipathic peptide with beta propensity, which was called ABZ12. Both peptides are able to surround the membrane protein and displace surfactant molecules. To our best knowledge, this is the first molecular mechanism proposed for "peptergency".

The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases. More precisely, genetic variants of the human UPF0016 ortholog transmembrane protein 165 (TMEM165) have been linked to congenital disorders of glycosylation (CDG). The Saccharomyces cerevisiae ortholog Gdt1p has been shown to be involved in calcium homeostasis and protein glycosylation. Moreover, plant and bacterial UPF0016 members appear to have putative roles in Mn 2+ homeostasis. Here, we produced the yeast UPF0016 member Gdt1p in the bacterial host Lactococcus lactis Using Mn 2+ -induced quenching of Fura-2-emitted fluorescence, we observed that Gdt1p mediates Mn 2+ influx, in addition to its previously reported regulation of Ca 2+ influx. The estimated K m values of Gdt1p of 15.6 ± 2.6 μm for Ca 2+ and 83.2 ± 9.8 μm for Mn 2+ indicated that Gdt1p has a higher affinity for Ca 2+ than for Mn 2+ In yeast cells, we found that Gdt1p is involved in the resistance to high Mn 2+ concentration and controls total Mn 2+ stores. Lastly, we demonstrated that GDT1 deletion affects the activity of the yeast Mn 2+ -dependent Sod2p superoxide dismutase, most likely by modulating cytosolic Mn 2+ concentrations. Taken together, we obtained first evidence that Gdt1p from yeast directly transports manganese, which strongly reinforces the suggested link between the UPF0016 family and Mn 2+ homeostasis and provides new insights into the molecular causes of human TMEM165-associated CDGs. Our results also shed light on how yeast cells may regulate Golgi intraluminal concentrations of manganese, a key cofactor of many enzymes involved in protein glycosylation., (© 2018 Thines et al.)

Membrane fission is essential to life. It is required for many fundamental cellular processes, as diverse as cyto- and karyokinesis, organelle division, membrane repair, and membrane trafficking and endocytosis. While membrane fission was originally seen as resulting from the action of mechanoenzymes such as dynamin, it is clear that the reality is more complex. In this review, we propose an updated overview of fission mechanisms, and try to extract essential requirements for each. We also present examples of cellular processes that involve these fission mechanisms. Finally, we list pending questions, whether they are specific to a peculiar fission mechanism or more general to the field., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca 2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated 17 mutations in the yeast Golgi-localized Ca 2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca 2+ -ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation., (© 2017 John Wiley & Sons Ltd.)

Symbiotic bacteria are common in insects and can affect various aspects of their hosts' biology. Although the effects of insect symbionts have been clarified for various insect symbiosis models, due to the difficulty of cultivating them in vitro , there is still limited knowledge available on the molecular features that drive symbiosis. Serratia symbiotica is one of the most common symbionts found in aphids. The recent findings of free-living strains that are considered as nascent partners of aphids provide the opportunity to examine the molecular mechanisms that a symbiont can deploy at the early stages of the symbiosis (i.e., symbiotic factors). In this work, a proteomic approach was used to establish a comprehensive proteome map of the free-living S. symbiotica strain CWBI-2.3 T . Most of the 720 proteins identified are related to housekeeping or primary metabolism. Of these, 76 were identified as candidate proteins possibly promoting host colonization. Our results provide strong evidence that S. symbiotica CWBI-2.3 T is well-armed for invading insect host tissues, and suggest that certain molecular features usually harbored by pathogenic bacteria are no longer present. This comprehensive proteome map provides a series of candidate genes for further studies to understand the molecular cross-talk between insects and symbiotic bacteria., Competing Interests: The authors declare there are no competing interests.

TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn 2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn 2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn 2+ -sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn 2+ -induced degradation of TMEM165., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F 1 F O -ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+) supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+) sensitivity was altered in TMEM165 depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+) homeostasis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Pmp3p-related proteins are highly conserved proteins that exist in bacteria, yeast, nematodes and plants, and its transcript is regulated in response to abiotic stresses, such as low temperature or high salinity. Pmp3p was originally identified in Saccharomyces cerevisiae, and it belongs to the sensitive to Na(+) (SNA)-protein family, which comprises four members--Pmp3p/Sna1p, Sna2p, Sna3p and Sna4p. Deletion of the PMP3 gene conferred sensitivity to cytotoxic cations, whereas removal of the other SNA genes did not lead to clear phenotypic effects. It has long been believed that Pmp3p-related proteins have a common and important role in the modulation of plasma membrane potential and in the regulation of intracellular ion homeostasis. Here, we show that several growth phenotypes linked to PMP3 deletion can be modulated by the removal of specific genes involved in sphingolipid synthesis. These genetic interactions, together with lipid binding assays and epifluorescence microscopy, as well as other biochemical experiments, suggest that Pmp3p could be part of a phosphoinositide-regulated stress sensor., (© 2015. Published by The Company of Biologists Ltd.)

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologues to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE both to confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The UPF0016 family is a group of uncharacterized membrane proteins, well conserved through evolution and defined by the presence of one or two copies of an E-Φ-G-D-(KR)-(ST) consensus motif. Our previous results have shown that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and might form a new group of cation/Ca2+ exchangers. Most members of the family are made of two homologous clusters of three transmembrane spans, separated by a central loop and assembled with an opposite orientation in the membrane. However, some bacterial members of the family have only one cluster of transmembrane domains. Among these 'single-domain membrane proteins' some cyanobacterial members were found as pairs of adjacent genes within the genome, but each gene was slightly different. We performed a bioinformatic analysis to propose the molecular evolution of the UPF0016 family and the emergence of the antiparallel topology. Our hypotheses were confirmed experimentally using functional complementation in yeast. This suggests an important and conserved function for UPF0016 proteins in a fundamental cellular process. We also show that members of the UPF0016 family share striking similarities, but no primary sequence homology, with members of the cation/Ca2+ exchangers (CaCA) superfamily. Such similarities could be an example of convergent evolution, supporting the previous hypothesis that members of the UPF0016 family are cation/Ca2+ exchangers.

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.

Acm2, the major autolysin of Lactobacillus plantarum, is a tripartite protein. Its catalytic domain is surrounded by an O-glycosylated N-terminal region rich in Ala, Ser, and Thr (AST domain), which is of low complexity and unknown function, and a C-terminal region composed of five SH3b peptidoglycan (PG) binding domains. Here, we investigate the contribution of these two accessory domains and of O-glycosylation to Acm2 functionality. We demonstrate that Acm2 is an N-acetylglucosaminidase and identify the pattern of O-glycosylation (21 mono-N-acetylglucosamines) of its AST domain. The O-glycosylation process is species-specific as Acm2 purified from Lactococcus lactis is not glycosylated. We therefore explored the functional role of O-glycosylation by purifying different truncated versions of Acm2 that were either glycosylated or non-glycosylated. We show that SH3b domains are able to bind PG and are responsible for Acm2 targeting to the septum of dividing cells, whereas the AST domain and its O-glycosylation are not involved in this process. Notably, our data reveal that the lack of O-glycosylation of the AST domain significantly increases Acm2 enzymatic activity, whereas removal of SH3b PG binding domains dramatically reduces this activity. Based on this antagonistic role, we propose a model in which access of the Acm2 catalytic domain to its substrate may be hindered by the AST domain where O-glycosylation changes its conformation and/or mediates interdomain interactions. To the best of our knowledge, this is the first time that O-glycosylation is shown to control the activity of a bacterial enzyme.

TMEM165 has recently been identified as a novel protein involved in CDG-II. TMEM165 has no biological function described so far. Different mutations were recently found in patients with Golgi glycosylation defects and harboring a peculiar skeletal phenotype. In this study, we examined the effect of naturally occurring mutations on the intracellular localization of TMEM165 and their abilities to complement the TMEM165-deficient yeast, gdt1▵. Wild-type TMEM165 was present within Golgi compartment, plasma membrane and late endosomes/lysosomes, whereas mutated TMEM165 were found differentially localized according to the mutations. We demonstrated that, in the yeast functional assay with TMEM165 ortholog Gdt1, the homozygous point mutation correlating with a mild phenotype restores the yeast functional assay, whereas the truncated mutation, associated with severe disease, failed to restore Gdt1 function. These studies highly suggest that these clinically relevant point mutations do not affect the protein function but critically changes the subcellular protein localization. Moreover, the data point to a critical role of the YNRL motif in TMEM165 subcellular localization.

Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Transmembrane protein 165 (TMEM165) belongs to an uncharacterized family of membrane proteins called Uncharacterized Protein Family 0016, which are well conserved throughout evolution and share characteristics reminiscent of the cation/Ca(2+) exchanger superfamily. Gcr1 dependent translation factor 1 (Gdt1p), the budding yeast member of this family, contributes to Ca(2+) homeostasis via an uncharacterized Ca(2+) transport pathway localized in the Golgi apparatus. The gdt1Δ mutant was found to be sensitive to high concentrations of Ca(2+), and interestingly, this sensitivity was suppressed by expression of TMEM165, the human ortholog of Gdt1p, indicating conservation of function among the members of this family. Patch-clamp analyses on human cells indicated that TMEM165 expression is linked to Ca(2+) ion transport. Furthermore, defects in TMEM165 affected both Ca(2+) and pH homeostasis. Based on these results, we propose that Gdt1p and TMEM165 could be members of a unique family of Golgi-localized Ca(2+)/H(+) antiporters and that modification of the Golgi Ca(2+) and pH balance could explain the glycosylation defects observed in TMEM165-deficient patients.

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.

Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

AtTSPO is a TspO/MBR domain-protein potentially involved in multiple stress regulation in Arabidopsis. As in most angiosperms, AtTSPO is encoded by a single, intronless gene. Expression of AtTSPO is tightly regulated both at the transcriptional and post-translational levels. It has been shown previously that overexpression of AtTSPO in plant cell can be detrimental, and the protein was detected in the endoplasmic reticulum (ER) and Golgi stacks, contrasting with previous findings and suggesting a mitochondrial subcellular localization for this protein. To ascertain these findings, immunocytochemistry and ABA induction were used to demonstrate that, in plant cells, physiological levels of AtTSPO colocalized with AtArf1, a mainly Golgi-localized protein in plant cells. In addition, fluorescent protein-tagged AtTSPO was targeted to the secretory pathway and did not colocalize with MitoTracker-labelled mitochondria. These results suggest that the polytopic membrane protein AtTSPO is cotranslationally targeted to the ER in plant cells and accumulates in the Trans-Golgi Network. Heterologous expression of AtTSPO in Saccharomyces cerevisiae, yeast devoid of TSPO-related protein, resulted in growth defects. However, subcellular fractionation and immunoprecipitation experiments showed that AtTSPO was targeted to mitochondria where it colocalized and interacted with the outer mitochondrial membrane porin VDAC1p, reminiscent of the subcellular localization and activity of mammalian translocator protein 18 kDa TSPO. The evolutionarily divergent AtTSPO appears therefore to be switching its sorting mode in a species-dependent manner, an uncommon peculiarity for a polytopic membrane protein in eukaryotic cells. These results are discussed in relation to the recognition and organelle targeting mechanisms of polytopic membrane proteins in eukaryotic cells.

The increasing popularity of proteomics has created a need for quantitative analysis methods. As a result, many different techniques are now available for performing gel-based or gel-free quantitative proteomic analysis of biological samples. These techniques provide an insight into global protein expression from identification to quantification, taking into account the highly dynamic status of the proteome. With its relatively small and compact genome, yeast is widely used as a model organism for molecular genetics, cell biology, and proteomic studies. In this review, we will show how quantitative proteomics has been used to study the effect of different stresses on yeast.

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI delta. We showed that recombinant CKI delta phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI delta correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI delta, strengthening the key role of this residue in the control of dCK activity. However, neither CKI delta inhibitors nor CKI delta siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI delta in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI delta could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme., (Copyright 2010 Elsevier Inc. All rights reserved.)

Split-inteins can be used to generate backbone cyclized peptide as a source of new bioactive molecules. In this work we show that cysteine-mediated splicing can be performed in the oxidative environment of the periplasm of Escherichia coli. Cyclization of the TEM-1 beta-lactamase and of small randomized peptides was demonstrated using an artificially permuted version of the DnaB mini-intein from Synechocystis sp. PCC6803 strain fused to a signal sequence. For small peptides, a signal sequence that promotes cotranslational translocation had to be used. Efficient backbone cyclization was observed for more than 50% of combinatorial peptides featuring a fully randomized sequence inserted between a serine and glycine that are necessary for fast splicing. Furthermore, by coexpressing a mutant of the pIV outer membrane pore protein of fd bacteriophage, we showed that peptides can diffuse in the extracellular medium. These results open new routes for searching compounds acting on new targets such as exported and membrane proteins or pathogen microorganisms.