Mitochondrial carriers are believed widely to be homodimers both in the inner membrane of the organelle and in detergents. The dimensions and molecular masses of the detergent and proteindetergent micelles were measured for yeast ADP͞ATP carriers in a range of different detergents. The radius of the carrier at the midpoint of the membrane, its average radius, its Stokes' radius, its molecular mass, and its excluded volume were determined. These parameters are consistent with the known structural model of the bovine ADP͞ATP carrier and they demonstrate that the yeast mitochondrial ADP͞ATP carriers are monomeric in detergents. Therefore, models of substrate transport have to be considered in which the carrier operates as a monomer rather than as a dimer.analytical ultracentrifugation ͉ membrane protein ͉ oligomeric state ͉ size-exclusion chromatography ͉ transport M itochondrial carriers transport nucleotides, amino acids, cofactors, carboxylic acids, and inorganic anions across the mitochondrial inner membrane to link metabolic pathways in the cytosol with those in the mitochondrial matrix. These transport steps are required for oxidative phosphorylation, the synthesis and degradation of amino acids and lipids, the synthesis of iron-sulfur clusters and heme, the synthesis of RNA, DNA, and proteins, and the generation of heat by dissipation of the proton electrochemical potential (1, 2).Mitochondrial carriers consist of three homologous amino acid sequence repeats of Ϸ100 amino acids (3). Each repeat is folded into two transmembrane ␣-helices and contains the signature motif P-X-[DE]-X-X- [RK]. The structural fold of the protein has threefold pseudosymmetry and consists of a barrel of six-transmembrane ␣-helices and three short helices parallel to the membrane plane (4-6). The prolines of the signature motif kink the odd-numbered ␣-helices sharply, and the charged residues form a salt bridge network, which closes the central cavity on the matrix side of the protein (5).The mitochondrial ADP͞ATP carrier and other mitochondrial carriers are believed to be homodimers. This view is based on inhibitor binding studies (7-9), small-angle neutron scattering (10), differential tagging (11), size-exclusion chromatography (12-14), equilibrium sedimentation (13, 14), native gel electrophoresis (12, 15, 16), cross-linking experiments (8, 17-19), tandem fusions (20-23), and cysteine replacement and spin labeling studies (24, 25). Yet, the carriers do not form an integrated 12 ␣-helical bundle that could explain their stability in detergents (4). Moreover, the monomer contains a potential pathway for translocation of substrates across the membrane (4-6), opening the possibility that the carrier functions as a monomer.We have investigated the oligomeric state of the yeast mitochondrial ADP͞ATP carriers AAC2 and AAC3, which are 87% identical in sequence, by determining their dimensions and molecular masses in a range of different detergents. These experiments indicate that the carriers are monomeric in mild detergents. ResultsSize...

Mitochondrial carriers are believed widely to be dimers both in structure and function. However, the structural fold is a barrel of six transmembrane ␣-helices without an obvious dimerisation interface. Here, we show by negative dominance studies that the yeast mitochondrial ADP/ATP carrier 2 from Saccharomyces cerevisiae (AAC2) is functional as a monomer in the mitochondrial membrane. Adenine nucleotide transport by wild-type AAC2 is inhibited by the sulfhydryl reagent 2-sulfonatoethyl-methanethiosulfonate (MTSES), whereas the activity of a mutant AAC2, devoid of cysteines, is unaffected. Wild-type and cysteine-less AAC2 were coexpressed in different molar ratios in yeast mitochondrial membranes. After addition of MTSES the residual transport activity correlated linearly with the fraction of cysteine-less carrier present in the membranes, and so the two versions functioned independently of each other. Also, the cysteine-less and wild-type carriers were purified separately, mixed in defined ratios and reconstituted into liposomes. Again, the residual transport activity in the presence of MTSES depended linearly on the amount of cysteine-less carrier. Thus, the entire transport cycle for ADP/ATP exchange is carried out by the monomer. mechanism ͉ membrane protein ͉ negative dominance studies ͉ oligomeric state ͉ transport M itochondrial carriers are believed widely to exist and function as homo-dimers (1-23). However, structural data are inconsistent with biochemical and biophysical studies that have been used to provide support for the existence of dimers in membranes and in detergents. First, the projection structure of the mitochondrial yeast ADP/ATP carrier demonstrated that the structural fold consists of six transmembrane (TM) ␣-helices rather than an intercalated bundle of twelve ␣-helices (24). Second, there are no extensive protein-protein interfaces between neighboring carriers in the crystals, as would be expected for proteins that require cooperative interaction to function (25). Third, the density distribution had 3-fold pseudo symmetry in agreement with the 3-fold sequence repeats found in all mitochondrial carriers (26,27). Fourth, the substrate translocation pathway appeared to be in the centre of the protein, rather than at the interface between two monomers (24). Fifth, although the proteins formed two-dimensional crystals of dimers in rows in both the P22 1 2 1 and P2 crystal forms (24), it was monomeric in detergent before crystal formation (28). Sixth, the atomic structure of the bovine ADP/ATP carrier in detergent determined by x-ray crystallography of P2 1 2 1 2 and C222 1 crystal forms showed that the structural fold was a six-␣-helical bundle with three additional short ␣-helices in the mitochondrial matrix (13). Seventh, the P2 1 2 1 2 crystal did not contain pairs of interacting proteins with the same orientation (13), but the C222 1 crystal contained two types of dimers, one of which has been proposed to be biologically significant (11,29). However, all of the protein-protein interacti...

Background:Major histocompatibility complex (MHC) class I chain-related protein A (MICA) and MHC class I chain-related protein B (MICB) are polymorphic proteins that are induced upon stress, damage or transformation of cells which act as a ‘kill me' signal through the natural-killer group 2, member D receptor expressed on cytotoxic lymphocytes. MICA/B are not thought to be constitutively expressed by healthy normal cells but expression has been reported for most tumour types. However, it is not clear how much of this protein is expressed on the cell surface.Methods:Using a novel, well-characterised antibody and both standard and confocal microscopy, we systematically profiled MICA/B expression in multiple human tumour and normal tissue.Results:High expression of MICA/B was detected in the majority of tumour tissues from multiple indications. Importantly, MICA/B proteins were predominantly localised intracellularly with only occasional evidence of cell membrane localisation. MICA/B expression was also demonstrated in most normal tissue epithelia and predominantly localised intracellularly. Crucially, we did not observe qualitative differences in cell surface expression between tumour and MICA/B expressing normal epithelia.Conclusions:This demonstrates for the first time that MICA/B is more broadly expressed in normal tissue and that expression is mainly intracellular with only a small fraction appearing on the cell surface of some epithelia and tumour cells.

To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy. The EC value of the mGITRL-FP was compared with an anti-GITR antibody in an agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors. The mGITRL-FP had an almost 50-fold higher EC value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8 and CD4 T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment. These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. .

Most mitochondrial carriers carry out equimolar exchange of substrates and they are believed widely to exist as homo-dimers. Here we show by differential tagging that the yeast mitochondrial ADP/ATP carrier AAC2 is a monomer in mild detergents. Carriers with and without six-histidine or hemagglutinin tags were co-expressed in defined molar ratios in yeast mitochondrial membranes. Their specific transport activity was unaffected by tagging or by co-expression. The co-expressed carriers were extracted from the membranes with mild detergents and purified rapidly by affinity chromatography. All of the untagged carriers were in the flow-through of the affinity column, whereas all of the tagged carriers bound to the column and were eluted subsequently, showing that stable dimers, consisting of associated tagged and untagged carriers, were not present. The specific inhibitors carboxyatractyloside and bongkrekic acid and the substrates ADP, ATP and ADP plus ATP were added during the experiments to determine whether lack of association might have been caused by carriers being prevented from cycling through the various states in the transport cycle where dimers might form. All of the protein was accounted for, but stable dimers were not detected in any of these conditions, showing that yeast ADP/ATP carriers are monomeric in detergents in agreement with their hydrodynamic properties and with their structure. Since strong interactions between monomers were not observed in any part of the transport cycle, it is highly unlikely that the carriers function cooperatively. Therefore, transport mechanisms need to be considered in which the carrier is operational as a monomer.