The dynamic dimerization of the yeast ADP/ATP carrier in the inner mitochondrial membrane is affected by conserved cysteine residues

The ADP/ATP carrier (AAC) that facilitates the translocation of ATP made in mitochondria is inserted at the inner mitochondrial membrane by the TIM10-TIM22 protein import system. Here we addressed the state of the AAC precursor during insertion (stage IV of import) and identified residues of the carrier important for dimerization. By a combination of (i) import of a mix of His-tagged and untagged versions of AAC either 35S-labeled or unlabeled, (ii) import of a tandem covalent dimer AAC into wild-type mitochondria, and (iii) import of monomeric AAC into mitochondria expressing only the tandem covalent dimer AAC, we found that the stage IV intermediate is a monomer, and this stage is probably the rate-limiting step of insertion in the membrane. Subsequent dimerization occurs extremely rapidly (within less than a minute). The incoming monomer dimerizes with monomeric endogenous AAC suggesting that the AAC dimer is very dynamic. Conserved Cys residues were found not to affect insertion significantly, but they are crucial for the dimerization process to obtain a functional carrier.     

The human mitochondrial ADP/ATP carriers: kinetic properties and biogenesis of wild-type and mutant proteins in the yeast S. cerevisiae

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.     

Subunits of the yeast mitochondrial ADP/ATP carrier: cooperation within the dimer

The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family (MCF). It exchanges ADP and ATP between matrix and intermembrane space. It is postulated from numerous experiments that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, and it is inferred that the active unit is a dimer, too. However, the structure of beef Ancp bound to CATR obtained at high resolution is that of a monomer. To ascertain the dimeric organization of Ancp, we have constructed covalent tandem dimers of which one "subunit" (protomer) is the wild type and the other is inactive for ADP/ATP exchange. We have chosen either the op1 mutant or another member of the MCF, the phosphate carrier (Picp). Activities of the chimeras were first evaluated in vivo. The Ancp/op1 constructs exchange the adenine nucleotides. The Anc/Pic chimeras are considered as bifunctional forms since they exchange ADP and ATP and transport P(i) within the same cells. We have then controlled the fact that the chimeras are stable in vivo and in vitro. Proteinase K digestion showed that both protomers of Ancp/op1 have similar organization in the membrane. Analyses of kinetic properties indicated that protomers of Ancp/op1 chimeras crosstalk during the nucleotide exchange unlike those of Anc/Pic. However, full inhibition of phosphate uptake by CATR, a very specific inhibitor of Ancp, strongly suggests that the native functional unit of Ancp, and thus of Picp, is a dimer.     

Routes to and from the plasma membrane: bulk flow versus signal mediated endocytosis

Transport of proteins via the secretory pathway is controlled by a combination of signal dependent cargo selection as well as unspecific bulk flow of membranes and aqueous lumen. Using the plant vacuolar sorting receptor as model for membrane spanning proteins, we have distinguished bulk flow from signal mediated protein targeting in biosynthetic and endocytic transport routes and investigated the influence of transmembrane domain length. More specifically, long transmembrane domains seem to prevent ER retention, either by stimulating export or preventing recycling from post ER compartments. Long transmembrane domains also seem to prevent endocytic bulk flow from the plasma membrane, but the presence of specific endocytosis signals overrules this in a dominant manner.     

Mutations in the Arabidopsis Peroxisomal ABC Transporter COMATOSE Allow Differentiation between Multiple Functions In Planta: Insights from an Allelic Series

COMATOSE (CTS), the Arabidopsis homologue of human Adrenoleukodystrophy protein (ALDP), is required for import of substrates for peroxisomal β-oxidation. A new allelic series and a homology model based on the bacterial ABC transporter, Sav1866, provide novel insights into structure-function relations of ABC subfamily D proteins. In contrast to ALDP, where the majority of mutations result in protein absence from the peroxisomal membrane, all CTS mutants produced stable protein. Mutation of conserved residues in the Walker A and B motifs in CTS nucleotide-binding domain (NBD) 1 resulted in a null phenotype but had little effect in NBD2, indicating that the NBDs are functionally distinct in vivo. Two alleles containing mutations in NBD1 outside the Walker motifs (E617K and C631Y) exhibited resistance to auxin precursors 2,4-dichlorophenoxybutyric acid (2,4-DB) and indole butyric acid (IBA) but were wild type in all other tests. The homology model predicted that the transmission interfaces are domain-swapped in CTS, and the differential effects of mutations in the conserved “EAA motif” of coupling helix 2 supported this prediction, consistent with distinct roles for each NBD. Our findings demonstrate that CTS functions can be separated by mutagenesis and the structural model provides a framework for interpretation of phenotypic data.     

Mechanisms and concepts paving the way towards a complete transport cycle of plant vacuolar sorting receptors

Delivery of proteins to the lytic vacuole in plants is a complex cascade of selective interactions that specifically excludes residents of the endoplasmic reticulum and secreted proteins. Vacuolar transport must be highly efficient to avoid mistargeting of hydrolytic enzymes to locations where they could be harmful. While plant vacuolar sorting signals have been well described for two decades, it is only during the last 5 years that a critical mass of data was gathered that begins to reveal how vacuolar sorting receptors (VSRs) may complete a full transport cycle. Yet, the field is far from reaching a consensus regarding the organelles that could be involved in vacuolar sorting, their potential biogenesis, and the ultimate recycling of membranes and protein machinery that maintain this pathway. This review will highlight the important landmarks in our understanding of VSR function and compare recent transport models that have been proposed so that an emerging picture of plant vacuolar sorting mechanisms can be drawn.     

Structural determinants of ligand imprinting: a molecular dynamics simulation study of subtilisin in aqueous and apolar solvents

The phenomenon known as "ligand imprinting" or "ligand-induced enzyme memory" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation.     

Golgi-Dependent Transport of Vacuolar Sorting Receptors Is Regulated by COPII,AP1, and AP4 Protein Complexes in Tobacco

The cycling of vacuolar sorting receptors (VSRs) between early and late secretory pathway compartments is regulated by signals in the cytosolic tail, but the exact pathway is controversial. Here, we show that receptor targeting in tobacco (Nicotiana tabacum) initially involves a canonical coat protein complex II–dependent endoplasmic reticulum-to-Golgi bulk flow route and that VSR–ligand interactions in the cis-Golgi play an important role in vacuolar sorting. We also show that a conserved Glu is required but not sufficient for rate-limiting YXXɸ-mediated receptor trafficking. Protein–protein interaction studies show that the VSR tail interacts with the μ-subunits of plant or mammalian clathrin adaptor complex AP1 and plant AP4 but not that of plant and mammalian AP2. Mutants causing a detour of full-length receptors via the cell surface invariantly cause the secretion of VSR ligands. Therefore, we propose that cycling via the plasma membrane is unlikely to play a role in biosynthetic vacuolar sorting under normal physiological conditions and that the conserved Ile-Met motif is mainly used to recover mistargeted receptors. This occurs via a fundamentally different pathway from the prevacuolar compartment that does not mediate recycling. The role of clathrin and clathrin-independent pathways in vacuolar targeting is discussed.     

The Arabidopsis Peroxisomal ABC Transporter,Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.     

Valine 181 is critical for the nucleotide exchange activity of human mitochondrial ADP/ATP carriers in yeast

We isolated yeast Saccharomyces cerevisiae cells transformed with one of the three human adenine nucleotide carrier genes (HANC) that exhibited higher growth capacity than previously observed. The HANC genes were isolated from these clones, and we identified two independent mutations of HANC that led to replacement of valine 181 located in the fourth transmembrane segment by methionine or phenylalanine. Tolerance of this position toward substitution with various amino acids was systematically investigated, and since HANC/V181M was among the most efficient in growth complementation, it was more extensively studied. Here we show that increased growth capacities were associated with higher ADP/ATP exchange activities and not with higher human carrier amount in yeast mitochondria. These results are discussed in the light of the bovine Ancp structure, that shares more than 90% amino acid identity with Hancps, and its interaction with the lipid environment.