The modification of the conserved GXXXG motif of the membrane-spanning segment of subunit g destabilizes the supramolecular species of yeast ATP synthase

The supernumerary subunit g is found in all mitochondrial ATP synthases. Most of the conserved amino acid residues are present in the membrane C-terminal part of the protein that contains a dimerization motif GXXXG. In yeast, alteration of this motif leads to the loss of subunit g and of supramolecular structures of the ATP synthase with concomitant appearance of anomalous mitochondrial morphologies. Disulfide bond formation involving an engineered cysteine in position 109 of subunit g and the endogenous cysteine 28 of subunit e promoted g + g, e + g, and e + e adducts, thus revealing the proximity in the mitochondrial membrane of several subunits e and g. Disulfide bond formation between two subunits g in mitochondria increased the stability of an oligomeric structure of the ATP synthase in digitonin extracts. These data suggest the participation of the dimerization motif of subunit g in the formation of supramolecular structures and is in favor of the existence of ATP synthase associations, in the inner mitochondrial membrane, whose masses are higher than those of ATP synthase dimers.     

The yeast F(1)F(0)-ATP synthase: analysis of the molecular organization of subunit g and the importance of a conserved GXXXG motif

The F(1)F(0)-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes. Dimerization of the ATP synthase involves the physical association of the neighboring membrane-embedded F(0)-sectors. In yeast, the F(0)-sector subunits g and e (Su g and Su e, respectively) play a key role in supporting the formation of ATP synthase dimers. In this study we have focused on Su g to gain a better understanding of the function and the molecular organization of this subunit within the ATP synthase complex. Su g proteins contain a GXXXG motif (G is glycine, and X is any amino acid) in their single transmembrane segment. GXXXG can be a dimerization motif that supports helix-helix interactions between neighboring transmembrane segments. We demonstrate here that the GXXXG motif is important for the function and in particular for the stability of Su g within the ATP synthase. Using site-directed mutagenesis and cross-linking approaches, we demonstrate that Su g and Su e interact, and our findings emphasize the importance of the membrane anchor regions of these proteins for their interaction. Su e also contains a conserved GXXXG motif in its membrane anchor. However, data presented here would suggest that an intact GXXXG motif in Su g is not essential for the Su g-Su e interaction. We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein.     

The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane.

A conserved putative dimerization GxxxG motif located in the unique membrane-spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues. These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion-like structures. By taking advantage of the presence of the endogenous cysteine 28 residue in the wild-type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts. The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers.     

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.     

Recognition of a basic AP-2 binding motif within the C2B domain of synaptotagmin is dependent on multimerization

Synaptotagmin is a multifunctional membrane protein that may regulate exo-endocytic cycling of synaptic vesicles at the presynaptic plasmalemma. Its C2B domain has been postulated to interact with a variety of effector molecules including acidic phospholipids, phosphoinositides, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), calcium channels, and the clathrin adaptor complex AP-2. Here we report that a basic motif within the C2B domain is required and sufficient for binding to AP-2 via its mu2 subunit and that this interaction is dependent on multimerization of the AP-2 binding site. Moreover, we show that upon fusion to a plasma membrane reporter protein this sequence is sufficient to target the chimeric molecule for internalization. We hypothesize that basic motifs within multimeric membrane proteins may represent a novel type of clathrin/AP-2-dependent endocytosis signal.     

Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

Tethering factors are organelle-specific multisubunit protein complexes that identify, along with Rab guanosine triphosphatases, transport vesicles and trigger their SNARE-mediated fusion of specific transport vesicles with the target membranes. Little is known about how tethering factors discriminate between different trafficking pathways, which may converge at the same organelle. In this paper, we describe a phosphorylation-based switch mechanism, which allows the homotypic vacuole fusion protein sorting effector subunit Vps41 to operate in two distinct fusion events, namely endosome-vacuole and AP-3 vesicle-vacuole fusion. Vps41 contains an amphipathic lipid-packing sensor (ALPS) motif, which recognizes highly curved membranes. At endosomes, this motif is inserted into the lipid bilayer and masks the binding motif for the δ subunit of the AP-3 complex, Apl5, without affecting the Vps41 function in endosome-vacuole fusion. At the much less curved vacuole, the ALPS motif becomes available for phosphorylation by the resident casein kinase Yck3. As a result, the Apl5-binding site is exposed and allows AP-3 vesicles to bind to Vps41, followed by specific fusion with the vacuolar membrane. This multifunctional tethering factor thus discriminates between trafficking routes by switching from a curvature-sensing to a coat recognition mode upon phosphorylation.     

Structure-guided identification of a new catalytic motif of oligosaccharyltransferase

Asn-glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn-X-Thr/Ser-motif-dependent manner. We also determined the 2.7-A resolution crystal structure of the C-terminal soluble domain of Pyrococcus STT3. The structure-based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well-conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipid-linked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide-asparagine bond.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

Identification of a conserved calmodulin-binding motif in the sequence of F0F1 ATPsynthase inhibitor protein

The natural inhibitor proteins IF1 regulate mitochondrial F0F1 ATPsynthase in a wide range of species. We characterized the interaction of CaM with purified bovine IF1, two bovine IF1 synthetic peptides, as well as two homologous proteins from yeast, namely IF1 and STF1. Fluorometric analyses showed that bovine and yeast inhibitors bind CaM with a 1:1 stoichiometry in the pH range between 5 and 8 and that CaM-IF1 interaction is Ca2+-dependent. Bovine and yeast IF1 have intermediate binding affinity for CaM, while the Kd (dissociation constant) of the STF1-CaM interaction is slightly higher. Binding studies of CaM with bovine IF1 synthetic peptides allowed us to identify bovine IF1 sequence 33-42 as the putative CaM-binding region. Sequence alignment revealed that this region contains a hydrophobic motif for CaM binding, highly conserved in both yeast IF1 and STF1 sequences. In addition, the same region in bovine IF1 has an IQ motif for CaM binding, conserved as an IQ-like motif in yeast IF1 but not in STF1. Based on the pH and Ca2+ dependence of IF1 interaction with CaM, we suggest that the complex can be formed outside mitochondria, where CaM could regulate IF1 trafficking or additional IF1 roles not yet clarified.     

Role of the conserved lysine within the Walker A motif of human DMC1

During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1_K132R) or alanine (hDMC1_K132A). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1_K132R and hDMC1_K132A variants were devoid of ATP hydrolysis. The hDMC1_K132R variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1_K132R variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1_K132A variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.     

The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

Ribonuclease P (RNase P) catalyzes the metal-dependent 5′ end maturation of precursor tRNAs (pre-tRNAs). In Bacteria, RNase P is composed of a catalytic RNA (PRNA) and a protein subunit (P protein) necessary for function in vivo. The P protein enhances pre-tRNA affinity, selectivity, and cleavage efficiency, as well as modulates the cation requirement for RNase P function. Bacterial P proteins share little sequence conservation although the protein structures are homologous. Here we combine site-directed mutagenesis, affinity measurements, and single turnover kinetics to demonstrate that two residues (R60 and R62) in the most highly conserved region of the P protein, the RNR motif (R60–R68 in Bacillus subtilis), stabilize PRNA complexes with both P protein (PRNA•P protein) and pre-tRNA (PRNA•P protein•pre-tRNA). Additionally, these data indicate that the RNR motif enhances a metal-stabilized conformational change in RNase P that accompanies substrate binding and is essential for efficient catalysis. Stabilization of this conformational change contributes to both the decreased metal requirement and the enhanced substrate recognition of the RNase P holoenzyme, illuminating the role of the most highly conserved region of P protein in the RNase P reaction pathway.     

Role of walker motif A of RuvB protein in promoting branch migration of holliday junctions. Walker motif a mutations affect Atp binding, Atp hydrolyzing, and DNA binding activities of Ruvb.

Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote branch migration of Holliday junctions during homologous recombination and recombinational repair. To elucidate the role of the Walker motif A of RuvB (GXGKT; X indicates a nonconserved residue) in ATP hydrolysis and branch migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys(68) and Thr(69). K68R, K68A, and T69A mutants except T69S failed to complement UV-sensitive phenotype of the ruvB strain. These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees. K68R, K68A, and T69A were defective in ATP hydrolysis and branch migration activities in vitro. In the presence of Mg(2+), K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction. K68A and T69A could form hexamers in the presence of Mg(2+) and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer. In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding. However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of K68R to DNA. All of the three mutant RuvBs could physically interact with RuvA. These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys(68) and Thr(69) residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the branch migration activity.     

The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity

DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs approximately 17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group approximately 17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis-Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.     

Identification of a mammalian homologue of the fungal Tom70 mitochondrial precursor protein import receptor as a thyroid hormone-regulated gene in specific brain regions

Thyroid hormone is an important regulator of mammalian brain maturation. By differential display PCR, we isolated a cDNA clone (S2) that is specifically up-regulated in the striatum of neonatal hypothyroid rats. S2 was identified as KIAA0719, the first human gene distantly homologous to the fungal Tom70, which encodes a member of the translocase mitochondrial outer membrane complex involved in the import of preproteins into the mitochondria. By northern and in situ hybridization studies, KIAA0719 was found to be up-regulated in the striatum, nucleus accumbens, and discrete cortical layers of 15-day-old hypothyroid rats. In contrast, lower expression was found in the olfactory tubercle, whereas no differences were detected in other brain regions. Significantly, treatment of hypothyroid animals with single injections of thyroxine restored the normal levels of KIAA0719 expression. Moreover, treatment of control animals with thyroxine led to a reduced expression, demonstrating a negative hormonal regulation in vivo. Thus, KIAA0719 gene expression is regulated by thyroid hormone in the neonatal rat brain in a region-specific fashion. Given the role of the homologous Tom70 gene, the alteration of KIAA0719 expression may contribute to the changes in mitochondrial morphology and physiology caused by hypothyroidism in the developing rat brain.     

Identification of highly conserved hydrophobic amino acid motif in deduced amino acid sequence of Elymus sibiricus L. mitochondrial S13 ribosomal protein

The nucleotide sequence of mitochondrial ribosomal protein rps13 gene from wild perennial grass Elymus sibiricus is presented. It was determined by the method of PCR amplification with specific oligonucleotide primers and the direct sequencing of the amplification product. The sequence of E. sibiricus mitochondrial gene for S13 predicts a hydrophobic ribosomal protein of 116 amino acids that shows strong similarity to those of wheat (99.7% identity) and maize (98%). The deduced amino acid sequence of S13 protein from E. sibiricus and homologous plant's (Zea mays, Daucus carota, Nicotiana tabacum, Marchantia polymorpha) and nonplant's (Escherichia coli) proteins shows the presence of hydrophobic amino acids' motif -L-X10-L-X10-M-X10-L-X10-L-. Slightly modified it can be found in many other ribosomal proteins. This conserved motif is presumed to be particularly important for association of the ribosomal S13 protein with other proteins in the small subunit of the mitochondrial ribosome.     

The C2 domain calcium-binding motif: structural and functional diversity.

The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.     

Solution structure of the PHD finger from the human KIAA1045 protein

Cross‐brace structural motifs are required as a scaffold to design artificial RING fingers (ARFs) that function as ubiquitin ligase (E3) in ubiquitination and have specific ubiquitin‐conjugating enzyme (E2)‐binding capabilities. The Simple Modular Architecture Research Tool database predicted the amino acid sequence 131–190 (KIAA1045ZF) of the human KIAA1045 protein as an unidentified structural region. Herein, the stoichiometry of zinc ions estimated spectrophotometrically by the metallochromic indicator revealed that the KIAA1045ZF motif binds to two zinc atoms. The structure of the KIAA1045ZF motif bound to the zinc atoms was elucidated at the atomic level by nuclear magnetic resonance. The actual structure of the KIAA1045ZF motif adopts a C₄HC₃‐type PHD fold belonging to the cross‐brace structural family. Therefore, the utilization of the KIAA1045ZF motif as a scaffold may lead to the creation of a novel ARF.     

SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phosphatase SHP-2 have similar binding specificities

Suppressor of cytokine signaling-3 (SOCS-3) and the protein tyrosine phosphatase SHP-2 both regulate signaling by cytokines of the interleukin-6 family, and this is dependent upon recruitment to tyrosine 757 in the shared cytokine receptor subunit gp130. To better explore the overlap in ligand binding specificities exhibited by these two signaling regulators, we have mapped the phosphopeptide binding preferences of the SH2 domains from SOCS-3 and SHP-2. Degenerate phosphopeptide libraries were screened against recombinantly produced SH2 domains to determine the sequences of optimal phosphopeptide ligands. We found that the consensus ligand binding motif for SOCS-3 was pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic-(H/V/I/Y), while the consensus motif for SHP-2 was pY-(S/T/A/V/I)-X-(V/I/L)-X-(W/F). We validated these data through the design of phosphopeptide ligands based on the consensus motifs and found that these bound to SOCS-3 and SHP-2 with high affinity. Finally, we have compared the affinity of SOCS-3 for binding to phosphopeptides representing putative docking sites in the gp130, leptin and erythropoietin receptors. While SOCS-3 binds with much higher affinity to a gp130 phosphopeptide than to phosphopeptides derived from the other receptors, multiple SOCS-3 binding sites are predicted to exist in the leptin and erythropoietin receptors which may compensate for weaker binding to individual sites.