Since detergent-resistant lipid rafts are involved in pathogen invasion, cholesterol homeostasis, angiogenesis, neurodegenerative diseases and signal transduction, protein identification in the rafts could provide important information to study their function. Here, we analyzed detergent-resistant raft proteins isolated from rat liver by capillary liquid chromatography-tandem mass spectrometry. Out of 196 proteins identified, 32% belonged to the raft or plasma membrane, 24% to mitochondrial, 20% to microsomal, 7% to miscellaneous, and 17% are unknown proteins. For example, membrane-bound receptors, trimeric GTP-binding proteins, ATP-binding cassette transporters, and glycosylphosphatidylinositol-anchored proteins were identified in this analysis. Unexpectedly, there were many mitochondrial proteins, raising a new issue for the presence of mitochondrial rafts or the localization of mitochondrial proteins into plasma membrane rafts. We confirmed that ATP synthase alpha and beta were expressed on the surface of the plasma membrane in HepG2 hepatocytes by immunofluorescence, cell surface biotinylation, and cellular fractionation. They had two distinct biochemical properties, detergent insolubility and low density, suggesting that the ATP synthase complex might be located in plasma membrane rafts as well as in the mitochondria.

The E3 ligase Ltn1 and the deubiquitylase Ubp3-Bre5 titrate the level of ribosomal subunit ubiquitylation and thereby set the rate of ribosomal protein degradation by ribophagy in response to nutrient supply and the level of protein translation.

In order to detect and identify ubiquitous lipid raft marker proteins, we isolated lipid rafts from different mouse organs, including the liver, lung, large brain, and kidney, and analyzed their proteins via 2-DE. Many protein spots were determined to be ubiquitous in all of the lipid rafts, and were annotated via LC and MS/MS. Twelve proteins were identified as ubiquitous raft proteins, and most of these were determined to be mitochondrial proteins, including mortalin, prohibitin, voltage-dependent anion channel, ATP synthase, NADH dehydrogenase, and ubiquinol-cytochrome c reductase. Via immunoblotting, these proteins were shown to exist in detergent-resistant lipid rafts prepared using different organ tissues. Since these oxidationreduction respiratory chains and ATP synthase complex were detected in detergent-resistant lipid raft fractions which had been isolated from the plasma membrane but not from the mitochondria, and found in the cell surface when determined by immunofluoresence and immunohistochemistry, we conclude that plasma membrane lipid rafts might contain oxidation-reduction respiratory chains and ATP synthase complex.

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Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.conformational heterogeneity | Ltn1/Listerin | RING E3 ubiquitin ligase | translational surveillance | neurodegenerative disease

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Purpose To determine the risk factors for lateral meniscus and root tears in patients with acute anterior cruciate ligament (ACL) injuries. Methods A total of 226 patients undergoing acute ACL reconstruction were included in the study sample. Exclusion criteria were revisions, fractures, chronic cases, and multiple ligament injuries, with the exception of medial collateral ligament (MCL) injuries. The patients were divided into groups based on the presence of lateral meniscus and root tears by arthroscopy. Binary logistic regression was used to analyze risk factors including age, sex, body mass index (BMI), injury mechanism (contact/non-contact), Segond fracture, side-to-side laxity, location of bone contusion, medial and lateral tibial and meniscal slope, mechanical axis angle, and grade of pivot shift. Results Overall lateral meniscus (LM) tears were identiied in 97 patients (42.9%), and LM root tears were found in 22 patients (9.7%). The risk of an LM tear in ACL-injured knees increased with bone contusion on LTP (odds ratio [OR], 3.5; 95% conidence interval [CI] 1.419-8.634; P = 0.007), steeper lateral tibial slope (OR, 1.133; 95% CI 1.003-1.28; P = 0.045), MCL injury (OR, 2.618; 95% CI 1.444-4.746; P = 0.002), and non-contact injury mechanism (OR, 3.132; 95% CI 1.446-6.785; P = 0.004) in logistic regression analysis. The risk of LM root tear in ACL-injured knees increased with high-grade pivot shift (OR, 9.127; 95% CI 2.821-29.525; P = 0.000) and steeper lateral tibial slope (OR, 1.293; 95% CI 1.061-1.576; P = 0.011). ConclusionThe increased risk of LM lesions in acute ACL-injured knees should be considered if signiicant risk factors including bone contusion on lateral compartments, MCL injury, and a steeper lateral tibial slope are present. Moreover, high-grade rotational injury with steeper lateral tibial slope are also signiicant risk factors for LM root tears, and therefore care should be taken by clinicians not to miss such lesions. Level of evidence III.

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