Transport of proteins across mitochondrial membranes

The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of preproteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c ₁ heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c ₁ preproteins, and the mitochondrial processing peptidase which cleaves signal sequence after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle. are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments — the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix — are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c ₁ heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c ₁ preproteins, and the mitochondrial processing peptidase which cleaves signal sequences after import of preproteins into the matrix. Thus, the study of transport of polypeptides through the mitochondrial membranes does not only contribute to the understanding of how biological membranes facilitate the penetration of macromolecules but also provides novel insights into the structure and function of this organelle.Abbreviations OM outer mitochondrial membrane - IM inner mitochondrial membrane - IMS mitochondrial intermembrane space - OMV outer membrane vesicles - AAC ADP/ATP carrier - PiC phosphate carrier - DHFR mouse cytosolic dihydrofolate reductase - F₁ subunit of F₁F₀ ^– ATPase - MPP mitochondrial processing peptidase     

The Charcot-Marie-Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system

Mitofusin-2 (Mfn2) is a mitochondrial membrane protein that participates in mitochondrial fusion in mammalian cells and mutations in the Mfn2 gene cause Charcot-Marie-Tooth neuropathy type 2A. Here, we show that Mfn2 loss-of-function inhibits pyruvate, glucose and fatty acid oxidation and reduces mitochondrial membrane potential, whereas Mfn2 gain-of-function increases glucose oxidation and mitochondrial membrane potential. As to the mechanisms involved, we have found that Mfn2 loss-of-function represses nuclear-encoded subunits of OXPHOS complexes I, II, III and V, whereas Mfn2 overexpression induced the subunits of complexes I, IV and V. Obesity-induced Mfn2 deficiency in rat skeletal muscle was also associated with a decrease in the subunits of complexes I, II, III and V. In addition, the effect of Mfn2 overexpression on mitochondrial metabolism was mimicked by a truncated Mfn2 mutant that is inactive as a mitochondrial fusion protein. Our results indicate that Mfn2 triggers mitochondrial energization, at least in part, by regulating OXPHOS expression through signals that are independent of its role as a mitochondrial fusion protein.     

Lactate, not pyruvate, is neuronal aerobic glycolysis end product: an in vitro electrophysiological study

For over 60 years, a distinction has been made between aerobic and anaerobic glycolysis based on their respective end products: pyruvate of the former, lactate of the latter. Recently we hypothesized that, in the brain, both aerobic and anaerobic glycolysis terminate with the formation of lactate from pyruvate by the enzyme lactate dehydrogenase (LDH). If this hypothesis is correct, lactate must be the mitochondrial substrate for oxidative energy metabolism via its oxidation to pyruvate, plausibly by a mitochondrial LDH. Here we employed electrophysiology of the rat hippocampal slice preparation to test and monitor the effects of malonate and oxamate, two different LDH inhibitors, and glutamate, a neuronal activator, in experiments, the results of which support the hypothesis that lactate, at least in this in vitro setting, is indeed the principal end product of neuronal aerobic glycolysis.     

Asymmetrically functional surface properties on biocompatible phospholipid polymer membrane for bioartificial kidney

To obtain a bioartificial kidney composed of a porous polymer membrane and renal cells, a polysulfone (PSf) membrane (PSM) blended with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer was prepared. The PSM flat membrane with a porous structure could be prepared from the polymer blend containing 1 wt % of the MPC polymer in PSf by the phase inversion technique in a dry-wet process. Asymmetrical surface properties were observed on both sides of the membrane surfaces. That is, the sponge layer formed at the substrate-contacting surface of the membrane had 10-20 microm pores, but the pores in the micrometer range could not be observed for a skin layer formed at the air-contacting surface of the membrane. At the sponge layer surface, the MPC unit composition was 7 times larger than that at the skin layer surface. The amount of proteins adsorbed on the surface corresponded to the MPC unit composition. On the skin layer, a small amount of adsorbed proteins and platelet adhesion could be suppressed compared with those on the sponge layer. However, the skin layer had a moderate protein adsorption, so it showed a sufficient cytocompatibility to enable renal tubule epithelial cells to adhere and proliferate in the membrane. Thus, it functioned well as a renal tubule. Therefore, because of both its hemocompatibility and cytocompatibility, we could conclude that the PSM membrane is useful for as a renal tubule device for a bioartificial kidney.     

Competition and protease sensitivity assays provide evidence for the existence of a hydrogenosomal protein import machinery in Trichomonas vaginalis

Hydrogenosomes are double membrane bounded redox organelles found in a number of amitochondriate protists and fungi. They are involved in carbohydrate metabolism and ATP synthesis and thus resemble mitochondria. Molecular analysis of the hydrogenosomal heat shock proteins Hsp70, Hsp60 and Hsp10 in Trichomonas vaginalis, one of the deepest-branching eukaryotes known to date, has revealed that these group exclusively with mitochondrial heat shock proteins. This finding indicates strongly that a progenitor organelle which gave rise to contemporary mitochondria and hydrogenosomes existed early in eukaryotic life. This hypothesis is further supported by similarities of hydrogenosomal and mitochondrial biogenesis. It was shown that T. vaginalis hydrogenosomal proteins are synthesized on free ribosomes in the cytosol with an N-terminal presequence that carries targeting information and is cleaved upon import into the organelle. Furthermore, as in mitochondrial import, hydrogenosomal protein import requires ATP, an electrochemical transmembrane potential and cytosolic protein factor(s). Here we demonstrate that inhibition of hydrogenosomal protein import occurs (i) in the presence of a synthetic presequence peptide and (ii) after pretreatment of hydrogenosomes with the protease trypsin. Trypsin pretreatment affects two hydrogenosomal membrane proteins of 31 and 70 kDa, respectively. Thus, we present evidence that import is saturable and that proteinaceous hydrogenosomal import receptor(s) exist. These results are a first step towards a characterization of the hydrogenosomal import machinery which should provide further insights into the relationship of hydrogenosomes and mitochondria and the evolution of protein targeting into organelles of endosymbiotic origin.     

Recent advances in the immunobiology of ceramide

Ceramide, a sphingosine-based lipid molecule, has emerged as a key regulator of a wide spectrum of biological processes such as cellular differentiation, proliferation, apoptosis and senescence. Sphingomyelinase-dependent hydrolysis of sphingomyelin and de novo synthesis involving the coordinated action of serinepalmitoyl transferase and ceramide synthase are the two major pathways involved in ceramide synthesis. Clustering of plasma membrane rafts into ceramide-enriched platforms serves as an important transmembrane signaling mechanism for cell surface receptors. Ceramides have been implicated in apoptosis, stress signaling cascades as well as ion channels. There is accumulating evidence that targeted manipulation of ceramide metabolism pathway has immense therapeutic potential and may eventually prove to be a boon in the design of novel strategies and development of innovative treatments for diverse conditions including cardiovascular diseases, cancer and Alzheimer's disease. As yet uncharacterized natural ceramide analogs and novel inhibitors of ceramide metabolism might prove to have potent effects in the drugs. In this review, we discuss significant advances that continue to provide intriguing insights into the complex cellular and molecular mechanisms underlying ceramide-mediated signaling cascades.     

The modulation of inter-organelle cross-talk to control apoptosis

Mitochondria fulfill a wide array of functions dedicated to the energetic metabolism as well as the control of cell death. These functions imply that mitochondria can be activated by a variety of signals and can integrate them to trigger a process called mitochondrial membrane permeabilization (MMP), which induces the ultimate events of apoptosis. MMP consists in a sudden increase in the permeability of mitochondrial membrane that results in the release of critical proapoptotic intermembrane space effectors into the cytosol such as cytochrome c, apoptosis-inducing factor (AIF), Smac/Diablo, Endo G, and pro-caspases. In many models of apoptosis, mitochondrial translocation of proteins and/or lipids concomitantly with alterations of the intracellular milieu has been shown to activate MMP. This applies to tumor suppressors of the Bax/Bcl-2 family (Bax, Bad, Bid, Bim), several protein kinases (Akt, ASK1, hexokinase), p53, NF-kappaB, and nuclear orphan receptors such as TR3/Nur77. After mitochondrial membrane association, these proteins target constitutive mitochondrial proteins including the permeability transition pore complex (PTPC), Bcl-X(L), HSP70, and/or the lipid interphase. Subsequently, they switch their vital function into a lethal function to promote membrane permeabilization and protein release. In this review, we will describe some general rules of inter-organelle cross-talk activating MMP and will review selected examples of pro-apoptotic protein translocation. Finally, we will propose new pharmacological strategies to modulate this process in a therapeutic perspective.     

Shiga toxin 1 induces apoptosis in the human myelogenous leukemia cell line THP-1 by a caspase-8-dependent, tumor necrosis factor receptor-independent mechanism

Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-alpha) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.     

Distinct types of protease systems are involved in homeostasis regulation of mitochondrial morphology via balanced fusion and fission

Mitochondrial morphology is dynamically regulated by fusion and fission. Several GTPase proteins control fusion and fission, and posttranslational modifications of these proteins are important for the regulation. However, it has not been clarified how the fusion and fission is balanced. Here, we report the molecular mechanism to regulate mitochondrial morphology in mammalian cells. Ablation of the mitochondrial fission, by repression of Drp1 or Mff, or by over‐expression of MiD49 or MiD51, results in a reduction in the fusion GTPase mitofusins (Mfn1 and Mfn2) in outer membrane and long form of OPA1 (L‐OPA1) in inner membrane. RNAi‐ or CRISPR‐induced ablation of Drp1 in HeLa cells enhanced the degradation of Mfns via the ubiquitin‐proteasome system (UPS). We further found that UPS‐related protein BAT3/BAG6, here we identified as Mfn2‐interacting protein, was implicated in the turnover of Mfns in the absence of mitochondrial fission. Ablation of the mitochondrial fission also enhanced the proteolytic cleavage of L‐OPA1 to soluble S‐OPA1, and the OPA1 processing was reversed by inhibition of the inner membrane protease OMA1 independent on the mitochondrial membrane potential. Our findings showed that the distinct degradation systems of the mitochondrial fusion proteins in different locations are enhanced in response to the mitochondrial morphology.     

The acetyl group deficit at the onset of contraction in ischaemic canine skeletal muscle

Considerable debate surrounds the identity of the precise cellular site(s) of inertia that limit the contribution of mitochondrial ATP resynthesis towards a step increase in workload at the onset of muscular contraction. By detailing the relationship between canine gracilis muscle energy metabolism and contractile function during constant-flow ischaemia, in the absence (control) and presence of pyruvate dehydrogenase complex activation by dichloroacetate, the present study examined whether there is a period at the onset of contraction when acetyl-coenzyme A (acetyl-CoA) availability limits mitochondrial ATP resynthesis, i.e. whether a limitation in mitochondrial acetyl group provision exists. Secondly, assuming it does exist, we also aimed to identify the mechanism by which dichloroacetate overcomes this 'acetyl group deficit ' . No increase in pyruvate dehydrogenase complex activation or acetyl group availability occurred during the first 20 s of contraction in the control condition, with strong trends for both acetyl-CoA and acetylcarnitine to actually decline (indicating the existence of an acetyl group deficit). Dichloroacetate increased resting pyruvate dehydrogenase complex activation, acetyl-CoA and acetylcarnitine by ≈20-fold (   P < 0.01  ), ≈3-fold (   P < 0.01  ) and ≈4-fold (   P < 0.01  ), respectively, and overcame the acetyl group deficit at the onset of contraction. As a consequence, the reliance upon non-oxidative ATP resynthesis was reduced by ≈40 % (   P < 0.01  ) and tension development was increased by ≈20 % (   P < 0.05  ) following 5 min of contraction. The present study has demonstrated, for the first time, the existence of an acetyl group deficit at the onset of contraction and has confirmed the metabolic and functional benefits to be gained from overcoming this inertia.     

Bcl-2: a prime regulator of mitochondrial redox metabolism in cancer cells

SIGNIFICANCE: Mitochondria play a critical role as death amplifiers during drug-induced apoptosis in cancer cells by providing pro-apoptotic factors that are released from the mitochondrial inter-membranous space upon the induction of mitochondrial outer membrane permeabilization. This intrinsic death signaling pathway is the preferred mechanism employed by most anticancer compounds, and as such, resistance to drug-induced apoptosis is invariably associated with inhibition of mitochondrial death signaling network. The latter is a function of a balance between the pro- and the anti-apoptotic members of the Bcl-2 family. Bcl-2 is the prototype anti-apoptotic protein that localizes to the mitochondria and blocks the recruitment and activation of pro-apoptotic proteins, such as Bax, to the mitochondria. RECENT ADVANCES AND CRITICAL ISSUES: Recent evidence has highlighted a novel mechanism of anti-apoptotic activity of Bcl-2 in addition to its canonical activity in regulating mitochondrial outer membrane permeabilization. This novel activity is a function of cellular redox regulation, in particular, mitochondrial metabolism in cancer cells. FUTURE DIRECTIONS: Here we review the current state of our understanding of the death inhibitory activity of Bcl-2 and provide insight into the novel functional biology of this remarkable protein, which could have implications for designing innovative strategies to overcome the problem of drug resistance in the clinical settings.     

Imaging and detecting molecular interactions of single transmembrane proteins

Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.     

Inhibition of proteasome activity promotes the correct localization of disease-causing alpha-sarcoglycan mutants in HEK-293 cells constitutively expressing beta-, gamma-, and delta-sarcoglycan

Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, alpha-, beta-, gamma-, and delta-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (beta, gamma, and delta) of the four sarcoglycans. In these betagammadelta-HEK cells, the lack of alpha-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of alpha-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of betagammadelta-HEK cells with disease-causing alpha-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies.     

Proteomic Analysis of Macrophages： A Potential Way to Identify Novel Proteins Associated with Activation of Macrophages for Tumor Cell Killing

One major mechanism through which macrophages effectively kill tumor cells requires cell to cell contact, indicating that certain molecules expressed on cell surface of activated macrophages may mediate the tumoricidal capability. Tumor necrosis factor （TNF） and nitric oxide （NO） are the two classical mediators of tumor cell death. However, evidence of discrepancy is accumulating indicating these known mediators do not appear to account for the broad and potent tumoricidal activity of macrophages. To obtain a full repertoire of tumoricidal activation-associated membrane proteins, we combined one-dimensional SDS-PAGE with capillary liquid chromatographytandem mass spectrometry （LC-MS/MS）. Using this technique, we identified 454 activated macrophage specifically expressed proteins with extremely high confidence, including most known activation markers of macrophages, such as NO synthase （iNOS）, Ym1, cyclooxygenase, etc. Membrane bound TNF-α was also identified on activated macrophages. However, it was also detected on thioglycolate elicited macrophages, indicating this molecule may not play a key role in conjugation-dependent tumor cell killing. In contrast, although NO has not been assigned as an effector molecule of conjugation-dependent tumoricidal pathway, iNOS was identified from membrane fraction of activated macrophages, suggesting NO may be involved in conjugation-dependent tumoricidal mechanism, because iNOS association with plasma membrane is ideally suited to deliver NO directly into the contacted tumor cells. This research provides not only new insights into macrophage conjugation-dependent tumoricidal mechanisms, but also a valuable data set of macrophage activation associated membrane proteins, thus providing better understanding of the functional mechanisms of macrophages in anti-tumor and other biological processes.     

The farnesyltransferase inhibitor manumycin A is a novel trypanocide with a complex mode of action including major effects on mitochondria

Eukaryotes modify numerous proteins, including small GTPases of the ras superfamily, with isoprenes as a mechanism for membrane attachment. Inhibition of farnesylation of ras has been successfully exploited to control cell growth, with promise in the clinic for treatment of human tumours. Using an in vitro screen of mammalian farnesyltransferase inhibitors, we have identified manumycin A as potently active against growth of both bloodstream and procyclic forms of Trypanosoma brucei. Other structural classes of farnesyltransferase inhibitors were far less effective. Exposure of T. brucei for brief periods to lethal concentrations of manumycin A resulted in subsequent cell death whilst the concentration required to achieve killing was dependent on serum concentration, suggesting partitioning of manumycin A into hydrophobic cellular sites. Manumycin A did not affect trypanosomal protein and DNA synthesis or cell cycle progression but altered incorporation of prenyl groups into several polypeptides indicating a specific effect on the prenylation without effect on other mevalonate pathway products, most importantly prenyl pyrophosphate levels. Morphological analysis indicated that manumycin A caused significant mitochondrial damage suggesting an additional site of action. Structural analogues of manumycin A containing a quinone were also highly trypanocidal and altered mitochondrial morphology, suggesting interference with electron/proton transport systems. Furthermore, manumycin A also elicited mitochondrial alterations in mammalian cells indicating that the effect is not confined to lower eukaryotes. Manumycin A is well tolerated in vivo but failed to cure experimental trypanosomiasis in mice.     

Radiation-induced oxidative DNA damage, 8-oxoguanine,in human peripheral T cells

The mechanism leading to the high level of radiosensitivity of T lymphocytes has not yet been fully described. In our previous study, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h after irradiation of 5 Gy, respectively. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c release was confirmed. In order to elucidate the mechanism which occurs prior to the mitochondrial membrane potential changes, we examined in the present study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we concluded that it remains necessary to evaluate the extent of radiation-induced oxidative DNA damage. Furthermore, it is important to analyze superoxide radical production and scavenging in terms of the variety of radiosensitivities found among various types of normal tissue cells and neoplastic cells.     

Contribution of FAT/CD36 to the regulation of skeletal muscle fatty acid oxidation: an overview

Long chain fatty acids (LCFAs) are an important substrate for ATP production within the skeletal muscle. The process of LCFA delivery from adipose tissue to muscle mitochondria involves many regulatory steps. Recently, it has been recognized that LCFA oxidation is not only dependent on LCFA delivery to the muscle, but also on regulatory steps within the muscle. Increasing selected fatty acid binding proteins/transporters on the plasma membrane facilitates a very rapid LCFA increase into the muscle, independent of any changes in LCFA delivery to the muscle. Such a mechanism of LCFA transporter translocation is activated by muscle contraction. Intramuscular triacylglycerols may also be hydrolysed to provide fatty acids for mitochondrial oxidation, particularly during exercise, when hormone-sensitive lipase and other enzymes are activated. Mitochondrial LCFA entry is also highly regulated. This however does not involve only the malonyl CoA carnitine palmitoyltransferase-I (CPTI) axis. Exercise-induced fatty acid entry into mitochondria is also regulated by at least one of the proteins (FAT/CD36) that also regulates plasma membrane fatty acid transport. Among individuals, differences in mitochondrial fatty acid oxidation appear to be correlated with the content of mitochondrial CPTI and FAT/CD36. This paper provides a brief overview of mechanisms that regulate LCFA uptake and oxidation in skeletal muscle during exercise and in obesity. We focus largely on our own work on FAT/CD36, which contributes to regulating, in a coordinated fashion, LCFA uptake across the plasma membrane and the mitochondrial membrane. Very little is known about the roles of FATP1-6 on fatty acid transport in skeletal muscle.     

Antigen Presentation by MHC Class II Molecules: Invariant Chain Function, Protein Trafficking, and the Molecular Basis of Diverse Determinant Capture

Major histocompatibility complex class II molecules are heterodimeric integral membrane proteins whose primary function is the presentation of antigenic peptides derived from proteins entering the endocytic pathway to CD4⁺ T lymphocytes. To accomplish this physiologic function, class II molecules must assemble in the secretory pathway without undergoing irreversible ligand association at that site, traffic efficiently to the endocytic pathway, and productively interact with protein ligands in these organelles before their ultimate expression on the plasma membrane. Here we review our work describing how invariant chain promotes the assembly and transport process, the complex itinerary of class II–invariant chain complexes through the endocytic pathway, the role of large protein fragments as substrates for class II binding, and the existence of a second pathway for antigen capture by mature class II molecules that complements that involving newly synthesized dimers. We integrate these observations into a coherent model for the operation of a class II-dependent antigen processing and presentation system able to capture diverse antigenic determinants present in proteins of varying structure.