The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state.

The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.This work was supported by the Medical Research Council Grant MC_UU_00015/1 (to E.R.S.K.), the Swiss National Science Foundation 31003A_179421/1 (to J-C.M.) and the Oncosuisse grant KFS-4434-02-2018 (to J-C.M.)

Beyond Conventional Boundaries: Advancing Equity and Inclusivity in Educational Supervision

This special issue of the Journal of Educational Supervision, Cases in Critical Supervision within School Contexts, critically examines educational supervision models, highlighting their limitations in fostering equity and inclusivity. As greater attention and awareness of diverse identities and experiences becomes more common in schools there also needs to be acknowledgement of how supervision is experienced by educators and enacted by instructional supervisors. We argue for a paradigm shift towards more empathetic and diverse supervisory practices that consider the varied backgrounds and needs of students. Throughout this special issue, researchers propose innovative approaches that prioritize collaboration, understanding, and the recognition of the unique potential of every teacher, educator, and student. Empowered with a willingness and an awareness of how educational experiences can be experienced differently, researchers suggest supervisory models to transform educational environments into spaces where all students, especially those from marginalized groups, can thrive

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.This work was funded by the intramural programme of the Medical Research Council (MRC). T.J.C was the recipient of an MRC graduate studentship

Mutation in VPS35 associated with Parkinson's disease impairs WASH complex association and inhibits autophagy

Endosomal protein sorting controls the localization of many physiologically important proteins and is linked to several neurodegenerative diseases. VPS35 is a component of the retromer complex, which mediates endosome-to-Golgi retrieval of membrane proteins such as the cation-independent mannose 6-phosphate receptor. Furthermore, retromer is also required for the endosomal recruitment of the actin nucleation promoting WASH complex. The VPS35 D620N mutation causes a rare form of autosomal-dominant Parkinson’s disease (PD). Here we show that this mutant associates poorly with the WASH complex and impairs WASH recruitment to endosomes. Autophagy is impaired in cells expressing PD-mutant VPS35 or lacking WASH. The autophagy defects can be explained, at least in part, by abnormal trafficking of the autophagy protein ATG9A. Thus, the PD-causing D620N mutation in VPS35 restricts WASH complex recruitment to endosomes, and reveals a novel role for the WASH complex in autophagosome formation

METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis.

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.Medical Research Council, UK [MC_UU_00015/4]; EMBO [ALFT 701-2013 to L.V.H]; ‘Fundação para a Ciência e a Tecnologia’ [PD/BD/105750/2014 to P.R.-G.]

EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation

<p>Abstract</p> <p>Background</p> <p>Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed <it>EFS </it>(<it>embryonal Fyn-associated substrate</it>) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated <it>EFS </it>promoter region CpG island in tumor classification and metastatic progression.</p> <p>Methods</p> <p><it>EFS </it>methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients.</p> <p>Results</p> <p>Analysis of 16 UM showed full methylation of the <it>EFS </it>CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with <it>EFS </it>methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with <it>EFS </it>methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of <it>EFS </it>mRNA expression with <it>EFS </it>methylation in UM. We further found that <it>EFS </it>methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. <it>EFS </it>methylation always affects both alleles in normal and tumor samples.</p> <p>Conclusions</p> <p>Biallelic <it>EFS </it>methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The <it>EFS </it>methylation of a UM may depend on which type of precursor cell the tumor originated from.</p

ISBS 2018 AUCKLAND CONFERENCE SPRINZ-HPSNZ-AUT MILLENNIUM APPLIED PROGRAMME

An interactive afternoon of sessions delivered by High Performance Sport New Zealand (HPSNZ) and AUT SPRINZ Biomechanists, Performance Analysts and other biomechanics relevant sport facing practitioners. The 11 sessions are at AUT Millennium (AUTM), which is a satellite site of AUT University and the Auckland training hub for many HPSNZ supported sports such as athletics, sailing, and swimming. These sports and others (cycling, rowing, snow sports etc.) will be represented in the line-up. The applied sessions involve practical demonstrations of aspects of analysis and/or tools used to deliver in the field to directly positively impact athletes performances on the world stage. Following these engaging sessions there will be tasting of New Zealand wine, allowing for further discussion and networking. Sir Graeme Avery will be acknowledged for his contribution to sport science. Mike Stanley is AUT Millennium Chief Executive & NZ Olympic Committee President will explain the partners in the facility. AUT Millennium is a charitable trust established to help New Zealanders live longer and healthier lives, and to enjoy and excel in sport through the provision of world-class facilities, services, research and education. Founded in 2002 as Millennium Institute of Sport and Health (MISH) by Sir Stephen Tindall and Sir Graeme Avery as a premium health and fitness facility for both athletes and the public alike. Partnered with AUT University in 2009, forming AUT Millennium, to expand research and education in the sporting sector. Professor Barry Wilson is an Adjunct Professor with SPRINZ at Auckland University of Technology and will be outlining the research and student opportunities. Martin Dowson is the General Manager Athlete Performance Support at High Performance Sport New Zealand and has overall responsibility for the programme. Simon Briscoe, AUT Millennium Applied Session Coordinator, is the head of the Performance and Technique Analysis discipline within HPSNZ. Simon is coordinating the applied sessions along with technical support from Dr Allan Carman, Research Fellow, AUT SPRINZ. Jodi Cossor and Matt Ingram will provide a demonstration of a multidisciplinary approach driven by biomechanical analysis for Paralympic swimmers. Justin Evans and Sarah-Kate Millar will provide a practical session assessing the athletes rowing stroke to assist the coach on technical changes. This session will demonstrate various rowing traits and how the biomechanist and coach can work together to optimise boat speed. Mike Schofield and Kim Hébert-Losier will provide a session looking at shotput and the evidence based approach to coaching. Dr Craig Harrison and Professor John Cronin will provide examples from the AUTM Athlete Development programme. Kim Simperingham and Jamie Douglas who work with high performance rugby athletes will outline sprinting mechanics in practice. Dr Bruce Hamilton, Fiona Mather, Justin Ralph and Rone Thompson will demonstrate the approach of HPSNZ and Cycling NZ performance health teams in the use of some specific tools for prevention of injury and optimisation of performance. Kelly Sheerin, Denny Wells and Associate Professor Thor Besier will provide examples of using IMU and motion capture methods for running and basketball biomechanics research, education and service. Dr Rodrigo Bini and Associate Professor Andrew Kilding will show how linking of biomechanics and physiology improves injury prevention and performance enhancement. Robert Tang, Andre de Jong and Farhan Tinwala discuss select projects developed by Goldmine, HPSNZ’s in-house engineering team, and how these innovations have enabled unprecedented levels of biomechanics feedback. Cameron Ross and Paul McAlpine demonstrate the technology being used at the Snow Sports NZ training centre in Cadrona to enhance load monitoring of athletes. This application allows greater insight into training performances and biomechanical loads than has been previously possible in the training environment. AUT Millennium tour guides are coordinated by Josh McGeown and include Enora Le Flao, Dustin Oranchuk, Erika Ikeda, Jono Neville, Aaron Uthoff, Andrew Pichardo, Farhan Tinwala, Shelley Diewald, Renata Bastos Gottgtroy, Jessica Yeoman, Casey Watkins, Eric Harbour, Anja Zoellner, Alyssa Joy Spence, Victor Lopez Jr, and Albert Chang

VARP is recruited on to endosomes by direct interaction with retromer, where together they function in export to the cell surface

VARP is a Rab32/38 effector that also binds to the endosomal/lysosomal R-SNARE VAMP7. VARP binding regulates VAMP7 participation in SNARE complex formation and can therefore influence VAMP7-mediated membrane fusion events. Mutant versions of VARP that cannot bind Rab32:GTP, designed on the basis of the VARP ankyrin repeat/Rab32:GTP complex structure described here, unexpectedly retain endosomal localization, showing that VARP recruitment is not dependent on Rab32 binding. We show that recruitment of VARP to the endosomal membrane is mediated by its direct interaction with VPS29, a subunit of the retromer complex, which is involved in trafficking from endosomes to the TGN and the cell surface. Transport of GLUT1 from endosomes to the cell surface requires VARP, VPS29, and VAMP7 and depends on the direct interaction between VPS29 and VARP. Finally, we propose that endocytic cycling of VAMP7 depends on its interaction with VARP and, consequently, also on retromer.G.G.H. was funded by a postdoctoral fellowship from the Canadian Institutes of Health Research; I.P.-D. was funded by a Federation of European Biochemical Societies Long-Term Fellowship; and L.W. was funded by an EMBO Long-Term Fellowship. I.P.D., I.B.S., and P.R.E. were funded by Medical Research Council (MRC) grant U105178845; L.P.J. and D.J.O. were funded by a Wellcome Trust fellowship; G.G.H., L.W., S.R.G., and J.P.L. were funded by an MRC programme grant (G0900113); O.B.Z. was funded by an Engineering and Physical Sciences Research Council-Doctoral Training Centre studentship; and M.E.H. and M.N.J.S. were funded by an MRC Senior Research Fellowship awarded to M.N.J.S. (G0701444). The Cambridge Institute for Medical Research is supported by a Wellcome Trust Strategic Award (100140).Peer reviewe