C-terminal Fusion Protein Research Articles

Molecular dynamics and in vitro analysis of Connexin43: A new 14‐3‐3 mode‐1 interacting protein

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.

Determination of the Substrate Specificities of N-Acetyl-d-glucosaminyltransferase

Heparan sulfate plays a wide range of physiological and pathological roles. Heparan sulfate consists of glucosamine and glucuronic/iduronic acid repeating disaccharides with various sulfations. Synthesis of structurally defined heparan sulfate oligosaccharides remains a challenge. Access to nonsulfated and unepimerized heparan sulfate backbone structures represents an essential step toward de novo enzymatic synthesis of heparan sulfate. The nonsulfated, unepimerized backbone heparan sulfate is similar to the capsular polysaccharide from Escherichia coli strain K5. The biosynthesis of this capsular polysaccharide involves in N-acetylglucosaminyltransferase (KfiA) and d-glucuronyltransferase (KfiC). In this study, we report the characterization of purified KfiA. KfiA was expressed in a C-terminal six-His fusion protein in BL21 star cells coexpressing chaperone proteins GroEL and GroES. The recombinant KfiA was purified to homogeneity with a Ni-agarose column. The binding affinities of various UDP-sugars for KfiA were determined using isothermal calorimetry titration, indicating that both the N-acetyl group and sugar type may be essential for donor substrates to bind KfiA. Kinetic analysis of KfiA toward different sizes of oligosaccharide revealed that KfiA is less sensitive to the size of the acceptor substrates. The results from this study open a new approach for the synthesis of the heparan sulfate backbone.

Cytoplasmic CstF-77 Protein Belongs to a Masking Complex with Cytoplasmic Polyadenylation Element-binding Protein in Xenopus Oocytes

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

Biochemical Characterization of VlmL, a Seryl-tRNA Synthetase Encoded by the Valanimycin Biosynthetic Gene Cluster

Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed.

Phosphorylation of Amphiphysin I by Minibrain Kinase/Dual-specificity Tyrosine Phosphorylation-regulated Kinase, a Kinase Implicated in Down Syndrome

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

The Mucin Muc4 Potentiates Neuregulin Signaling by Increasing the Cell-surface Populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.

Portal fusion protein constraints on function in DNA packaging of bacteriophage T4

Architecturally conserved viral portal dodecamers are central to capsid assembly and DNA packaging. To examine bacteriophage T4 portal functions, we constructed, expressed and assembled portal gene 20 fusion proteins. C-terminally fused (gp20-GFP, gp20-HOC) and N-terminally fused (GFP-gp20 and HOC-gp20) portal fusion proteins assembled in vivo into active phage. Phage assembled C-terminal fusion proteins were inaccessible to trypsin whereas assembled N-terminal fusions were accessible to trypsin, consistent with locations inside and outside the capsid respectively. Both N- and C-terminal fusions required coassembly into portals with approximately 50% wild-type (WT) or near WT-sized 20am truncated portal proteins to yield active phage. Trypsin digestion of HOC-gp20 portal fusion phage showed comparable protection of the HOC and gp20 portions of the proteolysed HOC-gp20 fusion, suggesting both proteins occupy protected capsid positions, at both the portal and the proximal HOC capsid-binding sites. The external portal location of the HOC portion of the HOC-gp20 fusion phage was confirmed by anti-HOC immuno-gold labelling studies that showed a gold 'necklace' around the phage capsid portal. Analysis of HOC-gp20-containing proheads showed increased HOC protein protection from trypsin degradation only after prohead expansion, indicating incorporation of HOC-gp20 portal fusion protein to protective proximal HOC-binding sites following this maturation. These proheads also showed no DNA packaging defect in vitro as compared with WT. Retention of function of phage and prohead portals with bulky internal (C-terminal) and external (N-terminal) fusion protein extensions, particularly of apparently capsid tethered portals, challenges the portal rotation requirement of some hypothetical DNA packaging mechanisms.

Differential Phosphoinositide Binding to Components of the G Protein-Gated K+ Channel

The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K(+) channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not.

The Carboxyl Terminus of the α-Subunit of the Amiloride-sensitive Epithelial Sodium Channel Binds to F-actin

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with K D values of 88 nM and 1.4 μM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct N ε-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.

Induction of Apoptosis by the Ste20-like Kinase SLK, a Germinal Center Kinase That Activates Apoptosis Signal-regulating Kinase and p38

Expression and activity of the germinal center kinase, Ste20-like kinase (SLK), are increased during kidney development and recovery from ischemic acute renal failure. In this study, we characterize the activation and functional role of SLK. SLK underwent dimerization via the C-terminal domain, and dimerization enhanced SLK activity. In contrast, the C-terminal domain of SLK did not dimerize with a related kinase, Mst1, and did not affect Mst1 activity. Phosphorylation/dephosphorylation of SLK were not associated with changes in kinase activity. SLK induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1) and increased ASK1 activity, indicating that ASK1 is a substrate of SLK. Moreover, SLK stimulated phosphorylation of p38 mitogen-activated protein kinase via ASK1, but not c-Jun N-terminal kinase nor extracellular signal-regulated kinase. Chemical anoxia and recovery during re-exposure to glucose (ischemia-reperfusion injury in cell culture) stimulated SLK activity. Overexpression of SLK enhanced anoxia/recovery-induced apoptosis, release of cytochrome c, and activities of caspase-8 and -9, and apoptosis was reduced significantly with p38 and caspase-9 inhibitors. Induction of the endoplasmic reticulum stress response by anoxia/recovery or tunicamycin (monitored by induction of Bip or Grp94 expression, phosphorylation of eukaryotic translation initiation factor 2alpha subunit, expression of CHOP, and activation of caspase-12) was attenuated in cells that overexpress SLK. Thus, SLK is an anoxia/recovery-dependent kinase that is activated via homodimerization and that signals via ASK1 and p38 to promote apoptosis. Attenuation of the protective aspects of the endoplasmic reticulum stress response by SLK may contribute to its proapoptotic effect.

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.

Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome.

Evidence for Direct Roles of Two Additional Factors, SECp43 and Soluble Liver Antigen, in the Selenoprotein Synthesis Machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.

Determination of the rate of muscarinic M 1 receptor plasma membrane delivery using a regulated secretion/aggregation system

Introduction In this study, we used the regulated secretion/aggregation technology (RPD™) to determine the rate of human muscarinic M 1 (hM 1) receptor plasma membrane delivery. Methods hM1 receptors were expressed in CHO cells as C-terminal fusion proteins to a conditional aggregation domain (CAD) consisting of four tandem mutant FKBP12 domains (F m). Results The CAD prevented the plasma membrane expression of hM1 receptors by causing the formation and intracellular retention of CAD-fused receptor aggregates as determined using intact cell [ 3H]NMS binding assays and epi-fluorescence microscopy, respectively. Aggregates of CAD-fused hM 1 receptor could be disrupted in a concentration-dependent manner by the F m-selective ligand AP21998, resulting in an increased hM 1 receptor plasma membrane expression. A furin cleavage site positioned between the CAD and the hM 1 receptor sequence was cleaved by furin once aggregates of fusion protein were disrupted by AP21998, thus ensuring their irreversible dissolution. The plasma membrane delivery of hM 1 receptors begins within 30 min of AP21998 exposure and the rate of delivery was constant for up to eight hours. In the continued presence of AP21998, hM 1 receptor plasma membrane expression continued to increase for up to 18 h, then began to decrease toward basal levels as incubation continued out to 72 h. Using mathematical models, we determined the rate constants for the plasma membrane delivery of hM 1 receptors from these data. Also, hM 1 receptors elicited phosphoinositide hydrolysis to carbachol once expressed at the plasma membrane and the pharmacology of the response varied depending upon the concentration of AP21998 used to cause plasma membrane expression. Discussion Overall, our data indicate that the RPD can be used to characterize the kinetics of receptor plasma membrane delivery and to characterize functional responses elicited to different numbers of plasma membrane expressed receptor.

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.

Topography of the Membrane Domain of the Liver Na+-Dependent Bile Acid Transporter

The ileal apical and liver basolateral bile acid transporters catalyze the Na+-dependent uptake of these amphipathic molecules in the intestine and liver. They contain nine predicted helical hydrophobic sequences (H1-H9) between the exoplasmic N-glycosylated N terminus and the cytoplasmic C terminus. Previous in vitro translation and in vivo alanine insertion scanning studies gave evidence for either nine or seven transmembrane segments, with H3 and H8 noninserted in the latter model. N-terminal GFP constructs containing either successive predicted segments or only the last two domains of the liver transporter following a membrane anchor signal were expressed in HEK-293 cells, and a C-terminal glycosylation flag allowed detection of membrane insertion. Western blot analysis with anti-GFP antibody after alkali and PNGase treatment showed that H1, H2, H3 behaved as competent transmembrane (TM) sequences. Results from longer constructs were difficult to interpret. H9, however, but not H8 was membrane-inserted. To analyze the intact transporter, a C-terminal YFP fusion protein was expressed as a functionally active protein in the plasma membrane of HEK-293 cells as seen by confocal microscopy. After limited tryptic digestion to ensure the accessibility of only exoplasmic lysine or arginine residues, molecular weight (MW) analysis of the five cleavage products on SDS-PAGE predicted the presence of seven transmembrane segments, H1, H2, H3, H4, H5, H6, and H9, with H7 and H8 exoplasmic. This new method provided evidence for seven membrane segments giving a new model of the membrane domain of this protein and probably the homologous ileal transporter, with H7/H8 as the transport region.

AtPEX2 and AtPEX10 Are Targeted to Peroxisomes Independently of Known Endoplasmic Reticulum Trafficking Routes

Controversy exists in the literature over the involvement of the endoplasmic reticulum (ER) in the delivery of membrane proteins to peroxisomes. In this study, the involvement of the ER in the trafficking of two Arabidopsis (Arabidopsis thaliana) peroxisomal membrane proteins was investigated using confocal laser scanning microscopy of living cells expressing fusions between enhanced yellow fluorescent protein (eYFP) and AtPEX2 and AtPEX10. The fusion proteins were always detected in peroxisomes and cytosol irrespective of the location of the eYFP tag or the level of expression. The cytosolic fluorescence was not due to cleavage of the eYFP reporter from the C-terminal fusion proteins. Blocking known ER transport routes using the fungal metabolite Brefeldin A or expressing dominant negative mutants of Sar1 or RabD2a had no effect on the trafficking of AtPEX2 and AtPEX10 to peroxisomes. We conclude that AtPEX2 and AtPEX10 are inserted into peroxisome membranes directly from the cytosol.

Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the β-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione–agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His 6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of α-helices, β-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.

A Novel, Evolutionarily Conserved Protein Phosphatase Complex Involved in Cisplatin Sensitivity

Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2).