Protease Sensitivity Research Articles

Endogenous protein mono-ADP-ribosylation in Arabidopsis thaliana

Protein mono-ADP-ribosylation post-translationally transfers the ADP-ribose moiety from the β-NAD+ donor to various protein acceptors. This type of modification has been widely characterized and shown to regulate protein activities in animals, yeast and prokaryotes, but has never been reported in plants. In this study, using [³²P]NAD+ as the substrate, ADP-ribosylated proteins in Arabidopsis were investigated. One protein substrate of 32 kDa in adult rosette leaves was found to be radiolabeled. Heat treatment, protease sensitivity and nucleotide derivative competition assays suggested a covalent reaction of NAD+ with the 32 kDa protein. [carbonyl-¹⁴C]NAD+ could not label the 32 kDa protein, confirming that the modification was ADP-ribosylation. Poly (ADP-ribose) polymerase inhibitor failed to suppress the reaction, but chemicals that destroy mono-ADP-ribosylation on specific amino acid residues could break up the linkage, suggesting that the reaction was not a poly-ADP-ribosylation but rather a mono-ADP-ribosylation. This modification mainly existed in leaves and was enhanced by oxidative stresses. In young seedlings, two more protein substrates with the size of 45 kDa and over 130 kDa, respectively, were observed in addition to the 32 kDa protein, indicating that different proteins were modified at different developmental stages. Although the substrate proteins remain to be identified, this is the first report on the characterization of endogenously mono-ADP-ribosylated proteins in plants.

X-ray Crystal Structure of the UCS Domain-Containing UNC-45 Myosin Chaperone from Drosophila melanogaster

UCS proteins, such as UNC-45, influence muscle contraction and other myosin-dependent motile processes. We report the first X-ray crystal structure of a UCS domain-containing protein, the UNC-45 myosin chaperone from Drosophila melanogaster (DmUNC-45). The structure reveals that the central and UCS domains form a contiguous arrangement of 17 consecutive helical layers that arrange themselves into five discrete armadillo repeat subdomains. Small-angle X-ray scattering data suggest that free DmUNC-45 adopts an elongated conformation and exhibits flexibility in solution. Protease sensitivity maps to a conserved loop that contacts the most carboxy-terminal UNC-45 armadillo repeat subdomain. Amino acid conservation across diverse UCS proteins maps to one face of this carboxy-terminal subdomain, and the majority of mutations that affect myosin-dependent cellular activities lie within or around this region. Our crystallographic, biophysical, and biochemical analyses suggest that DmUNC-45 function is afforded by its flexibility and by structural integrity of its UCS domain.

Protease Sensitivity of Corneodesmosin Variants Encoded by the Six More Common CDSN Haplotypes

Protease Sensitivity of Corneodesmosin Variants Encoded by the Six More Common CDSN Haplotypes

Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP

BackgroundAntimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens.Methodology and Principal FindingsAntibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH2 was effective against various “superbugs” including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection.Conclusions/SignificanceHydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise “tuning” of toxicity and proteolytic stability may be achieved by changing tag-length and adding W- or F-amino acid tags.

Tasa de mutaciones genotípicas y resistencia a antirretrovirales en un hospital general

Objectives The objective is to describe the resistance mutation rate in protease and reverse transcriptase genes and sensitivity to different antiretrovirals in our environment. Methods We performed an observational descriptive study in which we examined the samples provided at the clinical immunology laboratory between April 2004 and April 2009. We analysed both the resistance tests and the sensitivity to different drugs in patients with therapeutic failure using trugene hiv01 genotyping kits ®. Results We registered samples from 242 patients, 61 of which had no detectable resistance. The most prevalent mutations according to drug families were: for nucleoside analog reverse transcriptase inhibitors T215A/C/D/F/L/N/S/Y (24.10%), M184G/I/V/W (14.66%), M41J/L/R/T/W (11.24%) and K219E/G/H/N/R/T/W (10.24%). The highest levels of resistance corresponded to stavudine and lamivudine/emtricitabine, and tenofovir produced the least resistance in our environment. The non-analogues were K103N/R (23.98%), V179D/E/I/M/T (10.82%), A98E/G/S (10.53%) y K101E/P/Q/R (9.06%). Nevirapine presented greater resistance than efavirenz. Protease inhibitors were L10F/I/V (15.95%), M36I/L (13.81%), A71I/T/V (13.10%) and 154L/S/V (7.38%). The combination darunavir/ritonavir combination was that which presented the least resistance, and tipranavir/ritonavir and lopinavir/ritonavir the most resistance. Conclusions Antiretroviral resistance and sensitivity to retroviral treatment in our environment was similar to results from other studies in Spain, but differed in the high level of resistance to lamivudine/emtricitabine and lopinavir/ritonavir.

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na(+) channel (ENaC) mediates the rate-limiting step in transepithelial Na(+) transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

BackgroundSporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.Case presentationWe report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.ConclusionsA novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

D-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue

Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional (1)H NMR structure, and activity of D-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its D-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that D-maurocalcine should be an excellent vector for in vivo applications.

C.I.2 Defective glycosylation in congenital muscular dystrophies: From glycobiology to α-dystroglycanopathies

Glycosylation is the most common post-translational modification of proteins. The protein sequence data suggested that more than half of all proteins produced in mammalian cells are glycoproteins. Glycans of secreted glycoproteins affect many protein properties such as solubility, stability, protease sensitivity, and polarity, while glycans on cell surface glycoproteins are involved in various cellular functions including cell–cell and cell–matrix interactions during embryogenesis, immune reactions, and tumor development.

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.

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the alpha/beta-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the alpha/beta-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.

The 3-4 loop of an archaeal glutamate transporter homolog experiences ligand-induced structural changes and is essential for transport

Glutamatergic synaptic transmission is terminated by members of the excitatory amino acid transporter (EAAT) family of proteins that remove glutamate from the synaptic cleft by transporting it into surrounding glial cells. Recent structures of a bacterial homolog suggest that major motions within the transmembrane domain translocate the substrate across the membrane. However, the events leading to this large structural rearrangement are much less clear. Two reentrant loops have been proposed to act as extracellular and intracellular gates, but whether other regions of these proteins play a role in the transport process is unknown. We hypothesized that transport-related conformational changes could change the solvent accessibilities of affected residues, as reflected in protease sensitivity or small-molecule reactivity. In the model system Glt(Ph), an archaeal EAAT homologue from Pyrococcus horikoshii, limited trypsin proteolysis experiments initially identified a site in the long extracellular loop that stretches between helices 3 and 4 that becomes protected from proteolysis in the presence of a substrate, L-aspartate, or an inhibitor, DL-TBOA in the presence of Na(+), the cotransported ion. Using a combination of site-directed cysteine-scanning mutagenesis and fluorescein-5-maleimide labeling we found that positions throughout the loop experience these ligand-induced conformational changes. By selectively cleaving the 3-4 loop (via introduced Factor Xa sites) we demonstrate that it plays a vital role in the transport process; though structurally intact, the cleaved proteins are unable to transport aspartate. These results inculcate the 3-4 loop as an important player in the transport process, a finding not predicted by any of the available crystal structures of Glt(Ph).

Alteration of brain glycoproteins during aging

Glycosylation is the most common post-translational modification of proteins. Protein sequence data suggested that more than half of all proteins produced in mammalian cells are glycoproteins. Recent studies showed that glycans of secreted glycoproteins affect many protein properties, such as solubility, stability, protease sensitivity and polarity, whereas glycans on cell-surface glycoproteins are involved in various cellular functions, including cell-cell communication. Accordingly, the investigation of glycoprotein changes caused by aging is expected to help understand the aging process and to elucidate age-associated diseases. The present review will summarize our current knowledge of changes found in brain glycoproteins resulting from the aging process.

A Highly Toxic Cellular Prion Protein Induces a Novel, Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

DNA Binding by the ETS Protein TEL (ETV6) Is Regulated by Autoinhibition and Self-association

The ETS protein TEL, a transcriptional repressor, contains a PNT domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT domain and DNA-binding ETS domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.

Glucose regulates enzymatic sources of mitochondrial NADPH in skeletal muscle cells; a novel role for glucose‐6‐phosphate dehydrogenase

Reduced nicotinamide adenine dinucleotide (NADPH) is a functionally important metabolite required to support numerous cellular processes. However, despite the identification of numerous NADPH-producing enzymes, the mechanisms underlying how the organellar pools of NADPH are maintained remain elusive. Here, we have identified glucose-6-phosphate dehydrogenase (G6PDH) as an important source of NADPH in mitochondria. Activity analysis, submitochondrial fractionation, fluorescence microscopy, and protease sensitivity assays revealed that G6PDH is localized to the mitochondrial matrix. 6-ANAM, a specific G6PDH inhibitor, depleted mitochondrial NADPH pools and increased oxidative stress revealing the importance of G6PDH in NADPH maintenance. We also show that glucose availability and differences in metabolic state modulate the enzymatic sources of NADPH in mitochondria. Indeed, cells cultured in high glucose (HG) not only adopted a glycolytic phenotype but also relied heavily on matrix-associated G6PDH as a source of NADPH. In contrast, cells exposed to low-glucose (LG) concentrations, which displayed increased oxygen consumption, mitochondrial metabolic efficiency, and decreased glycolysis, relied predominantly on isocitrate dehydrogenase (ICDH) as the principal NADPH-producing enzyme in the mitochondria. Culturing glycolytic cells in LG for 48 h decreased G6PDH and increased ICDH protein levels in the mitochondria, further pointing to the regulatory role of glucose. 2-Deoxyglucose treatment also prevented the increase of mitochondrial G6PDH in response to HG. The role of glucose in regulating enzymatic sources of mitochondrial NADPH pool maintenance was confirmed using human myotubes from obese adults with a history of type 2 diabetes mellitus (post-T2DM). Myotubes from post-T2DM participants failed to increase mitochondrial G6PDH in response to HG in contrast to mitochondria in myotubes from control participants (non-T2DM). Hence, we not only identified a matrix-associated G6PDH but also provide evidence that metabolic state/glucose availability modulate enzymatic sources of NADPH.

Effect of mutations of the human serpin protein corticosteroid-binding globulin on cortisol-binding, thermal and protease sensitivity

Corticosteroid-binding globulin (CBG, transcortin) belongs to the serpin family of serine protease inhibitors (SERPINA6) and is mainly secreted by the liver. The negative acute phase protein CBG regulates free cortisol levels in the blood and distributes cortisol to its target tissues. So far no CBG serpin partner protease has been identified. However, its cleavage by human neutrophil elastase destroys ligand binding capacity and supposedly liberates cortisol at sites of inflammation. Here we report on the recombinant expression and secretion of human wild-type CBG and several novel mutants by human 293-EBNA cells. Functional characterization of wild-type and mutant CBG revealed distinct differences in ligand binding sensitivity to heat or elastase. Certain mutants are almost devoid of cortisol binding activity (Q232R and CBG Lyon), some display higher sensitivity for heat inactivation (G335V, Q232R and CBG Lyon) or for elastase cleavage (G335V). CBG mutant T342A is more resistant to elastase cleavage. Our data support the validity of the serpin structural concept. The expression system used provides functionally active human recombinant transcortin for further functional characterization of wild-type and human CBG mutant variants, which have been associated with altered serum free cortisol levels or pathophysiological constellations such as increased body weight, fatigue or hypotension.

Structural Hierarchy of Regulatory Elements in the Folding and Transport of an Intestinal Multidomain Protein

Human intestinal lactase-phlorizin hydrolase, LPH, encompasses four homologous domains, which presumably have evolved from two subsequent duplications of one ancestral gene. The profragment, LPHalpha, comprises homologous domains I and II and functions as an intramolecular chaperone in the context of the brush-border LPHbeta region of LPH. Here, we analyze the inter-relationship between homologous domains III and IV of LPHbeta and their implication in the overall structure, function, and trafficking of LPH. In silico analyses revealed potential domain boundaries for these domains as a basis for loop-out mutagenesis and construction of deletion or individual domain forms of LPH. Removal of domain IV, which contains lactase, results in a diminished phlorizin hydrolase activity, lack of dimerization in the endoplasmic reticulum (ER), but accelerated transport kinetics from the ER to the Golgi apparatus. By contrast, deletion of domain III, which harbors phlorizin hydrolase, generates a malfolded protein that is blocked in the ER. Interestingly, homologous domain III is transport-competent per se and sorted to the apical membrane in polarized Madin-Darby canine kidney cells. Nevertheless, it neither dimerizes nor acquires complete phlorizin hydrolase activity. Our data present a hierarchical model of LPH in which the homologous domain III constitutes (i) a fully autonomous core domain within LPH and (ii) another intramolecular chaperone besides the profragment LPHalpha. Nevertheless, the regulation of the trafficking kinetics and activity of domain III and entire LPH including elevation of the enzymatic activities require the correct dimerization of LPH in the ER, an event that is accomplished by the non-autonomous domain IV.

Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA(C)) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA(C). The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA(C) consists of at least three subdomains, a flexible linker, FlhA(CN), and FlhA(CC), and that FlhA(CN) becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA(C) not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.