Mutations in the V-ATPase Assembly Factor VMA21 Cause a Congenital Disorder of Glycosylation With Autophagic Liver Disease.

BACKGROUND AND AIMS: Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy. APPROACH AND RESULTS: Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways. CONCLUSIONS: Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

Differences in Acceleration and High-Intensity Activities Between Small-Sided Games and Peak Periods of Official Matches in Elite Soccer Players.

ABSTRACT: Dalen, T, Sandmæl, S, Stevens, TGA, Hjelde, GH, Kjøsnes, TN, and Wisløff, U. Differences in acceleration and high-intensity activities between small-sided games and peak periods of official matches in elite soccer players. J Strength Cond Res 35(7): 2018-2024, 2021-The purpose of this study was to compare whether the physical performance of players during 4 vs. 4 + goalkeeper (4 vs. 4) and 6 vs. 6 + goalkeeper (6 vs. 6) small-sided games (SSGs) is equivalent to those experienced during the most intense 5-minute period of soccer match play. Twenty-six male soccer players from an elite Norwegian league team took part. Players were monitored during 18 matches, 56 SSGs: twenty-eight 4 vs. 4 and twenty-eight 6 vs. 6 games. The ZXY Sport Tracking System was used to measure for each player the total distance covered, high-intensity running distance, sprint distance, number of accelerations, and player load (all expressed per minute). To compare the physical performance variables on players during the SSGs formats and match play, a 1-way analysis of variance with repeated measures was used. Players performed the same number of accelerations and player load in 4 vs. 4 (1.7 and 248, respectively) as in peak match (1.6 and 227, respectively), whereas in 6 vs. 6, there were 63% fewer accelerations and 15% lower player load (1.2 and 198, respectively), than in peak match. High-intensity running and sprint distance were significantly lower than mean match values in both 4 vs. 4 (4.1 and 0.2 m vs. 8.2 and 1.7 m) and 6 vs. 6 games (2.7 and 0.21 m vs. 8.2 and 1.7 m) (p < 0.05). In conclusion, only 4 vs. 4 SSGs are highly valuable, and in that, they elicit player load and accelerations to a level that is (at least) equivalent with peak periods of official match play.

Evolution of increased complexity in a molecular machine.

Many cellular processes are carried out by molecular 'machines'-assemblies of multiple differentiated proteins that physically interact to execute biological functions. Despite much speculation, strong evidence of the mechanisms by which these assemblies evolved is lacking. Here we use ancestral gene resurrection and manipulative genetic experiments to determine how the complexity of an essential molecular machine--the hexameric transmembrane ring of the eukaryotic V-ATPase proton pump--increased hundreds of millions of years ago. We show that the ring of Fungi, which is composed of three paralogous proteins, evolved from a more ancient two-paralogue complex because of a gene duplication that was followed by loss in each daughter copy of specific interfaces by which it interacts with other ring proteins. These losses were complementary, so both copies became obligate components with restricted spatial roles in the complex. Reintroducing a single historical mutation from each paralogue lineage into the resurrected ancestral proteins is sufficient to recapitulate their asymmetric degeneration and trigger the requirement for the more elaborate three-component ring. Our experiments show that increased complexity in an essential molecular machine evolved because of simple, high-probability evolutionary processes, without the apparent evolution of novel functions. They point to a plausible mechanism for the evolution of complexity in other multi-paralogue protein complexes.

Validity and reliability of 6-a-side small-sided game locomotor performance in assessing physical fitness in football players.

In order to determine whether small-sided game (SSG) locomotor performance can serve as a fitness indicator, we (1) compared 6-a-side (6v6) SSG-intensity of players varying in fitness and skill, (2) examined the relationship of the 6v6-SSG and Yo-Yo IR2 and (3) assessed the reliability of the 6v6-SSG. Thirty-three professional senior, 30 professional youth, 62 amateur and 16 professional woman football players performed 4 × 7 min 6v6-SSGs recorded by a Local Position Measurement system. A substantial subgroup (N = 113) also performed the Yo-Yo IR2. Forty-seven amateur players performed two or three 6v6-SSGs. No differences in 6v6-SSG time-motion variables were found between professional senior and professional youth players. Amateurs showed lower values than professional seniors on almost all time-motion variables (ES = 0.59-1.19). Women displayed lower high-intensity time-motion variables than all other subgroups. Total distance run during 6v6-SSG was only moderately related to Yo-Yo IR2 distance (r = 0.45), but estimated metabolic power, high speed (>14.4 km · h(-1)), high acceleration (>2 m · s(-2)), high power (>20 W · kg(-1)) and very high (35 W · kg(-1)) power showed higher correlations (r = 0.59-0.70) with Yo-Yo IR2 distance. Intraclass correlation coefficient values were higher for total distance (0.84) than other time-motion variables (0.74‒0.78). Although total distance and metabolic power during 6v6-SSG showed good reproducibility (coefficient of variation (CV) < 5%), CV was higher (8-14%) for all high-intensity time-motion variables. It was therefore concluded that standardised SSG locomotor performance cannot serve used as a valid and reliable fitness indicator for individual players.

Sorting of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase): identification of a necessary and sufficient Golgi/endosomal retention signal in Stv1p.

Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p). To identify residues responsible for sorting of the Golgi isoform of the V-ATPase, a random mutagenesis was performed on the N terminus of Stv1p. Subsequent characterization of mutant alleles led to the identification of a short peptide sequence, W(83)KY, that is necessary for proper Stv1p localization. Based on three-dimensional homology modeling to the Meiothermus ruber subunit I, we propose a structural model of the intact Stv1p-containing V-ATPase demonstrating the accessibility of the W(83)KY sequence to retrograde sorting machinery. Finally, we characterized the sorting signal within the context of a reconstructed Stv1p ancestor (Anc.Stv1). This evolutionary intermediate includes an endogenous W(83)KY sorting motif and is sufficient to compete with sorting of the native yeast Stv1p V-ATPase isoform. These data define a novel sorting signal that is both necessary and sufficient for trafficking of the V-ATPase within the Golgi/endosomal network.

Design of an accuracy study for position detection in football.

Only a few studies have analysed the accuracy of position detection systems. All of them analysed distances and velocities, which are the outcome of calculations based on x,y positions. The objective of this study was to analyse the accuracy of dynamic x,y position measurement of a radar- and an image-based system in football. One and two runners performed five different runs on four different locations on a football pitch. X,y positions recorded by the radar-based and the image-based system were compared to x,y positions measured by a Laveg laser device. Accuracies were analysed by means of root mean square error (RMSE). Results showed that the radar-based system detects positions more accurately than the image-based system (P = 0.000). Mean RMSE of the radar-based system was 0.24 m, RMSE of the image-based system was 0.73 m. The image-based system was significantly influenced by the location of the run, whereas the radar-based system was not. Based on the results of this study, we conclude that the radar-based system is more valid in detecting x,y positions than the image-based system. Future studies should take advantage of the new test design which allows more precise statements on the accuracy of a position detection system.

An early start at a professional soccer academy is no prerequisite for world cup soccer participation.

Introduction: 829 players from 32 nations on five continents participated in the 2022 men's World Cup tournament in Qatar. Not much is known about the youth careers of World Cup players from all over the world, especially about the age at which they began playing youth soccer in a professional academy. This study aimed to provide insights in the age national team players participating in World Cup Qatar 2022 started playing for a professional soccer academy and whether their starting age relates to continent and their current playing position (i.e., goalkeepers, defenders, midfielders, and forwards). Method: Systematic online desk research was conducted to determine the age at which World Cup players started playing for professional youth soccer organizations. The median and interquartile ranges were expressed for the starting age in professional youth soccer organizations and the current age at the World Cup. The variables were compared with playing position, the continent of the player's World Cup nation, and the continent on which the player was raised. Results: The results reveal that World Cup Qatar 2022 players started playing for professional soccer academies at a median age of 13.2 years (range: 4.2-22.6). In Europe, players started playing for professional youth soccer organizations earlier than players on other continents [χ2 (4) = 142.0, p < 0.001]. We also found a younger starting age in forwards than goalkeepers (p < 0.05). Discussion: In most established soccer nations in Europe and South-America, World Cup players started playing for professional soccer academies before the age of 12. However, a significant number of players started later, especially players on other continents, which reveals the different pathways youth players can follow to the elites.

The origin of suspended particulate matter in the Great Barrier Reef.

River run-off has long been regarded as the largest source of organic-rich suspended particulate matter (SPM) in the Great Barrier Reef (GBR), contributing to high turbidity, pollutant exposure and increasing vulnerability of coral reef to climate change. However, the terrestrial versus marine origin of the SPM in the GBR is uncertain. Here we provide multiple lines of evidence (13C NMR, isotopic and genetic fingerprints) to unravel that a considerable proportion of the terrestrially-derived SPM is degraded in the riverine and estuarine mixing zones before it is transported further offshore. The fingerprints of SPM in the marine environment were completely different from those of terrestrial origin but more consistent with that formed by marine phytoplankton. This result indicates that the SPM in the GBR may not have terrestrial origin but produced locally in the marine environment, which has significant implications on developing better-targeted management practices for improving water quality in the GBR.

SEMAT--the next generation of inexpensive marine environmental monitoring and measurement systems.

There is an increasing need for environmental measurement systems to further science and thereby lead to improved policies for sustainable management. Marine environments are particularly hostile and extremely difficult for deploying sensitive measurement systems. As a consequence the need for data is greatest in marine environments, particularly in the developing economies/regions. Expense is typically the most significant limiting factor in the number of measurement systems that can be deployed, although technical complexity and the consequent high level of technical skill required for deployment and servicing runs a close second. This paper describes the Smart Environmental Monitoring and Analysis Technologies (SEMAT) project and the present development of the SEMAT technology. SEMAT is a "smart" wireless sensor network that uses a commodity-based approach for selecting technologies most appropriate to the scientifically driven marine research and monitoring domain/field. This approach allows for significantly cheaper environmental observation systems that cover a larger geographical area and can therefore collect more representative data. We describe SEMAT's goals, which include: (1) The ability to adapt and evolve; (2) Underwater wireless communications; (3) Short-range wireless power transmission; (4) Plug and play components; (5) Minimal deployment expertise; (6) Near real-time analysis tools; and (7) Intelligent sensors. This paper illustrates how the capacity of the system has been improved over three iterations towards realising these goals. The result is an inexpensive and flexible system that is ideal for short-term deployments in shallow coastal and other aquatic environments.

Arabidopsis has two functional orthologs of the yeast V-ATPase assembly factor Vma21p.

How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.

ATP6AP2 functions as a V-ATPase assembly factor in the endoplasmic reticulum.

ATP6AP2 (also known as the [pro]renin receptor) is a type I transmembrane protein that can be cleaved into two fragments in the Golgi apparatus. While in Drosophila ATP6AP2 functions in the planar cell polarity (PCP) pathway, recent human genetic studies have suggested that ATP6AP2 could participate in the assembly of the V-ATPase in the endoplasmic reticulum (ER). Using a yeast model, we show here that the V-ATPase assembly factor Voa1 can functionally be replaced by Drosophila ATP6AP2. This rescue is even more efficient when coexpressing its binding partner ATP6AP1, indicating that these two proteins together fulfill Voa1 functions in higher organisms. Structure-function analyses in both yeast and Drosophila show that proteolytic cleavage is dispensable, while C-terminus-dependent ER retrieval is required for ATP6AP2 function. Accordingly, we demonstrate that both overexpression and lack of ATP6AP2 causes ER stress in Drosophila wing cells and that the induction of ER stress is sufficient to cause PCP phenotypes. In summary, our results suggest that full-length ATP6AP2 contributes to the assembly of the V-ATPase proton pore and that impairment of this function affects ER homeostasis and PCP signaling.

Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p.

Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN. vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.

Ykt6p is a multifunctional yeast R-SNARE that is required for multiple membrane transport pathways to the vacuole.

Intracellular membrane fusion requires that membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins on both vesicle and target membranes form a highly specific complex necessary to bring the membranes close in space. Ykt6p is a yeast R-SNARE protein that has been implicated in retrograde transport to the cis-Golgi compartment. Ykt6p has been also been found to fractionate with vacuole membranes and participate in a vacuolar SNARE complex in homotypic vacuole fusion. To investigate the role of Ykt6p in membrane traffic to the vacuole we generated temperature-sensitive mutations in YKT6. One mutation produces an early Golgi block to secretion, and overexpression of the SNARE protein Sft1p suppresses the growth and secretion defects of this mutation. These results are consistent with Ykt6p and Sft1p participating in a SNARE complex associated with retrograde transport to the cis-Golgi. A second set of mutations in YKT6 specifically affects post-Golgi membrane traffic to the vacuole, and the effects of these mutations are not suppressed by Sft1p overexpression. Defects are seen in carboxypeptidase Y sorting, alkaline phosphatase transport, and aminopeptidase I delivery, and in one mutant, overexpression of the SNARE protein Nyv1p suppresses the alkaline phosphatase transport defect. By mutationally separating early and late requirements for Ykt6p, our findings have revealed that Ykt6p is a R-SNARE protein that functions directly in the three biosynthetic pathways to the vacuole.

Role of Vma21p in assembly and transport of the yeast vacuolar ATPase.

The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V1) responsible for ATP hydrolysis and an integral membrane sector (V0) required for proton translocation. Biogenesis of V0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Delta cells, the major proteolipid subunit of V0 failed to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V0 subunits. Analysis of vmaDelta strains showed that binding of V0 subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V0 subunits, indicating that assembly of V0 occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V0 was transient and that Vma21p/V0 dissociation was concomitant with V0/V1 assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V0 and abrogated assembly of V0/V1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V0 in the vacuole, this interaction did not affect the assembly of vacuolar V0/V1 complexes. We conclude that Vma21p is not involved in regulating the interaction between V0 and V1 sectors, but that it has a crucial role in coordinating the assembly of V0 subunits and in escorting the assembled V0 complex into ER-derived transport vesicles.

Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae.

The late Golgi compartment is a major protein sorting station in the cell. Secreted proteins, cell surface proteins, and proteins destined for endosomes or lysosomes must be sorted from one another at this compartment and targeted to their correct destinations. The molecular details of protein trafficking pathways from the late Golgi to the endosomal system are becoming increasingly well understood due in part to information obtained by genetic analysis of yeast. It is now clear that proteins identified in yeast have functional homologues (orthologues) in higher organisms. We will review the molecular mechanisms of protein targeting from the late Golgi to endosomes and to the vacuole (the equivalent of the mammalian lysosome) of the budding yeast Saccharomyces cerevisiae.