Large Hydrophobic Amino Acids Research Articles

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD2)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys65 residue and its network of hydrogen bonds with the polar residues Ser45, Thr67, and Ser81, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys65 residue is activated by its interaction with Ser45 and Thr67 and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.

Specific Interaction between Tomato HsfA1 and HsfA2 Creates Hetero-oligomeric Superactivator Complexes for Synergistic Activation of Heat Stress Gene Expression

In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.

Insights into the Enzyme-Substrate Interaction in the Norovirus 3C-like Protease

The Glu54 residue of the norovirus 3C-like protease was implicated in proteolysis as a third-member carboxylate of the catalytic triad. The E54L mutant protease cleaved the sequence (133)LSFE/AP between the 3B and 3C regions of norovirus polyprotein, but did not cleave the sequence (198)ATSE/GK between the 3A and 3B. The 3BC junction mutation (3B-L133A or 3B-F135S) hampered the cleavage by the E54L protease, whereas the 3AB junction mutation (3A-A198L, S200F) allowed the E54L protease to digest. These results indicate that the E54L mutant protease is a substrate-specificity mutant and requires large hydrophobic amino acid residues at both P4 and P2 positions of the substrate. It was notable that the 3A-S200F P2 position mutation caused tight interaction between the wild-type protease and the C-terminus of the 3A protein, hence a decreased release rate of the product from the enzyme. This tight binding was dependent on the hydrophobicity of amino acid residues introduced at position 200 of the 3A region and was affected by the mutation in the bII-cII loop of the protease or the mutation of position 198 of 3A corresponding to the P4 position of the substrate. These results suggest that the protease and the substrate sense each other in the process of the proteolysis, being supported by crystal structures.

Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: Functional divergence of the dilysine ER retrieval motif in plant cells

Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the committed step in the production of glycerolipids, which are major components of cellular membranes, seed storage oils, and epicuticular wax coatings. While the biochemical activities of GPATs have been characterized in detail, the cellular features of these enzymes are only beginning to emerge. Here we characterized the phylogenetic relationships and cellular properties of two GPAT enzymes from the relatively large Arabidopsis thaliana GPAT family, including GPAT8, which is involved in cutin biosynthesis, and GPAT9, which is a new putative GPAT that has extensive homology with a GPAT from mammalian cells involved in storage oil formation and, thus, may have a similar role in plants. Immunofluorescence microscopy of transiently-expressed myc-epitope-tagged GPAT8 and GPAT9 revealed that both proteins were localized to the endoplasmic reticulum (ER), and differential permeabilization experiments indicated that their N- and C-termini were oriented towards the cytosol. However, these two proteins contained distinct types of ER retrieval signals, with GPAT8 possessing a divergent type of dilysine motif (-KK-COOH rather than the prototypic -KKXX-COOH or -KXKXX-COOH motif) and GPAT9 possessing a hydrophobic pentapeptide motif (-phi-X-X-K/R/D/E-phi-; where phi are large hydrophobic amino acid residues). Notably, the divergent dilysine motif in GPAT8 only functioned effectively when additional upstream residues were included to provide the proper protein context. Extensive mutational analyses of the divergent dilysine motif, based upon sequences present in the C-termini of other GPAT8s from various plant species, further expanded the functional definition of this molecular targeting signal, thereby providing insight to the targeting signals in other GPAT family members as well as other ER-resident membrane proteins within plant cells.

Novel Proteomics Strategy Brings Insight into the Prevalence of SUMO-2 Target Sites

Small ubiquitin-like modifier (SUMO) is covalently conjugated to its target proteins thereby altering their activity. The mammalian SUMO protein family includes four members (SUMO-1-4) of which SUMO-2 and SUMO-3 are conjugated in a stress-inducible manner. The vast majority of known SUMO substrates are recognized by the single SUMO E2-conjugating enzyme Ubc9 binding to a consensus tetrapeptide (PsiKXE where Psi stands for a large hydrophobic amino acid) or extended motifs that contain phosphorylated or negatively charged amino acids called PDSM (phosphorylation-dependent sumoylation motif) and NDSM (negatively charged amino acid-dependent sumoylation motif), respectively. We identified 382 SUMO-2 targets using a novel method based on SUMO protease treatment that improves separation of SUMO substrates on SDS-PAGE before LC-ESI-MS/MS. We also implemented a software SUMOFI (SUMO motif finder) to facilitate identification of motifs for SUMO substrates from a user-provided set of proteins and to classify the substrates according to the type of SUMO-targeting consensus site. Surprisingly more than half of the substrates lacked any known consensus site, suggesting that numerous SUMO substrates are recognized by a yet unknown consensus site-independent mechanism. Gene ontology analysis revealed that substrates in distinct functional categories display strikingly different prevalences of NDSM sites. Given that different types of motifs are bound by Ubc9 using alternative mechanisms, our data suggest that the preference of SUMO-2 targeting mechanism depends on the biological function of the substrate.

Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse—High specificity indicates high substrate selectivity

Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the β-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1′ distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2′ aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1′ and P2′ positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice.

Characterization of Carboxypeptidase A6, an Extracellular Matrix Peptidase

Carboxypeptidase A6 (CPA6) is a member of the M14 metallocarboxypeptidase family that is highly expressed in the adult mouse olfactory bulb and broadly expressed in embryonic brain and other tissues. A disruption in the human CPA6 gene is linked to Duane syndrome, a defect in the abducens nerve/lateral rectus muscle connection. In this study the cellular distribution, processing, and substrate specificity of human CPA6 were investigated. The 50-kDa pro-CPA6 is routed through the constitutive secretory pathway, processed by furin or a furin-like enzyme into the 37-kDa active form, and secreted into the extracellular matrix. CPA6 cleaves the C-terminal residue from a range of substrates, including small synthetic substrates, larger peptides, and proteins. CPA6 has a preference for large hydrophobic C-terminal amino acids as well as histidine. Peptides with a penultimate glycine or proline are very poorly cleaved. Several neuropeptides were found to be processed by CPA6, including Met- and Leu-enkephalin, angiotensin I, and neurotensin. Whereas CPA6 converts enkephalin and neurotensin into forms known to be inactive toward their receptors, CPA6 converts inactive angiotensin I into the biologically active angiotensin II. Taken together, these data suggest a role for CPA6 in the regulation of neuropeptides in the extracellular environment within the olfactory bulb and other parts of the brain.

Small Ubiquitin-like Modifier Modification Regulates the DNA Binding Activity of Glial Cell Missing Drosophila Homolog a

Glial cell missing Drosophila homolog a (GCMa) is an essential transcription factor for placental development, which controls the differentiation of the syncytiotrophoblast layer. Although the activity of GCMa can be post-translationally regulated by protein phosphorylation, ubiquitination, and acetylation, it is unknown whether GCMa activity can be regulated by sumoylation. In this report, we investigated the role of sumoylation in the regulation of GCMa activity. We demonstrated that Ubc9, the E2 component of the sumoylation machinery, specifically interacts with the N-terminal domain of GCMa and promotes GCMa sumoylation on lysine 156. Moreover, GCMa-mediated transcriptional activation was repressed by sumoylation but was enhanced in the presence of the SUMO-specific protease, SENP1. The repressive effect of sumoylation on GCMa transcriptional activity was attributed to decreased DNA binding activity of GCMa. Furthermore, structural analysis revealed a steric clash between the SUMO1 moiety of sumoylated GCMa and the DNA-binding surfaces of GCMa, which may destabilize the interaction between GCMa and its cognate DNA sequence. Our study demonstrates that GCMa is a new sumoylation substrate and its activity is down-regulated by sumoylation.

Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus

The ability of the immune system to distinguish between self and non-self is critical to the functioning of the immune response. A breakdown in these mechanisms can lead to the onset of autoimmune disease. Clinical and molecular data suggest that shared immunogenetic mechanisms lead to the autoimmune process. The most studied part of the autoimmune process is the human leukocyte antigen (HLA) region. Recently, progress has been made in narrowing down HLA cluster classifications based on structural and functional features of HLA alleles. Using this approach we have investigated 175 patients with hepatitis C virus (HCV)-induced type II cryoglobulinemia (MC), and compared them to a control group of 14,923 bone marrow donors. Additionally, we investigated the frequency of HLA homozygosity in the same groups of subjects. Our results provide evidence of a role for DR5 and DQ3 HLA class II clusters and a higher frequency of HLA homozygous leading to the clinical outcome of type II mixed cryoglobulinemic autoimmune disease. The DR5 cluster is characterized by a Glu in β9 and its polymorphism is connected with preferred anchors at β9 of the binding peptide, while the DQ3 cluster is characterized by Glu B86 and Leu B87, which allows the binding of large hydrophobic amino acids at p1 of the binding peptide. The mechanisms by which variations in HLA lead to autoimmunity remain unknown, although they are likely to be mediated by continuous presentation of HCV epitopes to T cells and a genetic background that limits the effective clearance of HCV. The results presented in this paper have increased our knowledge of the mechanism of autoimmune disease and B-cell lymphoproliferation during HCV infection. The work was performed in accordance with the principles of the 1983 Declaration of Helsinki. There is no conflict of interest.

The Inner Loop of Tetraspanins CD82 and CD81 Mediates Interactions with Human T Cell Lymphotrophic Virus Type 1 Gag Protein

The tetraspanin superfamily proteins play important roles in organizing membrane protein complexes, modulating integrin function, and controlling T cell adhesion. Tetraspanins such as CD82 contain two extracellular loops with its N terminus, C terminus, and inner loop exposed to the cytoplasm. The matrix (MA) domain of human T cell lymphotrophic virus, type 1 (HTLV-1), Gag interacts with the cytoplasmic face of the plasma membrane and is concentrated at tetraspanin-enriched microdomains. To understand the basis of this association, we generated site-directed mutations in the various domains of CD82 and used coimmunoprecipitation and colocalization approaches to examine interactions with HTLV-1 MA. The large extracellular loop of CD82, which is important for interactions with integrins, was not required for the association with HTLV-1 MA. The cytoplasmic N terminus and C terminus of CD82 were also dispensable for CD82-MA interactions. In contrast, mutations of conserved amino acids in the inner loop of CD82 or of palmitoylated cysteines that flank the inner loop diminished CD82 association with MA. HTLV-1 MA also interacted with the inner loop of CD81. Thus, association of HTLV-1 Gag with tetraspanin-enriched microdomains is mediated by the inner loops of CD81 and CD82.

Prorenin and Its Ancient Receptor

Prorenin, the precursor of renin, has for a long time been considered to be functionally inert, despite high circulating concentrations. Plasma levels of prorenin usually exceed renin levels and are even further increased under pathological conditions, such as in diabetes. First support of a functional significance of prorenin came from studies in transgenic rats, which overexpress a renin gene in the liver resulting in high circulating prorenin levels.1 Without systemic renin–angiotensin system activation and with normal blood pressure, these animals developed a vascular phenotype reminiscent of malignant hypertension. Moreover, in a series of elegant experiments, Ichihara et al2–4 supported the concept of prorenin as a relevant factor in the pathophysiology of diseases, such as hypertension and diabetes, by using a peptide that binds and inhibits the activation of the precursor. Thus, this peptide attenuated nephropathy in streptozotocin-induced diabetes2 and cardiac fibrosis in stroke-prone spontaneously hypertensive rats.3 By successfully repeating the streptozotocin experiment in mice deficient for the angiotensin II type 1a (AT1a) receptor, they reported recently that these effects are independent of renin activation or angiotensin II generation and concluded that the peptide, in fact, inhibits the interaction of prorenin with a receptor.4 In this issue of Hypertension , Saris et al5 contribute further evidence for an angiotensin-independent action of prorenin on cells by showing that neonatal cardiomyocytes respond to purified prorenin by p38 mitogen-activated protein (MAP) kinase phosphorylation and broad changes in gene expression partially also caused by the MAP kinase activation. Because the authors had shown before that these cells are not able to synthesize angiotensin II after the addition of prorenin, they consider the observed effects as being …

The influence of l-amino acid molecular structure on the phase transition temperature of hydroxypropyl methylcellulose

The influence of molecular structure of small ionised molecules on the thermo-reversible sol:gel phase transition of a hydroxyalkyl cellulose ether has been investigated with respect to an l-amino acid series. The concentration dependency of the phase transition temperature of 1% w/w hydroxypropyl methylcellulose solutions in the presence of different amino acids was studied turbidimetrically. The effects of different amino acids ranged from −33.54 to +45.52 °C M −1. Correlations between the effect on phase transition temperature and amino acid molecular properties showed the strongest relationships with amino acid hydrophobicity. Small hydrophilic amino acids lowered the transition temperature, whereas large hydrophobic aromatic amino acids increased this temperature. The effect of different amino acids was proposed to be a balance between the ability of their hydrophilic regions to dehydrate and disrupt the polymer hydration sheath, and the ability of their hydrophobic regions to associate with and solubilise the methoxyl-dominated regions of the polymer.

FcγRIIB Ile232Thr transmembrane polymorphism associated with human systemic lupus erythematosus decreases affinity to lipid rafts and attenuates inhibitory effects on B cell receptor signaling

The B cell inhibitory receptor FcgammaRIIB plays crucial roles in the maintenance of self-tolerance. We have identified a polymorphism FCGR2B c.695T>C that results in the non-conservative replacement of 232Ile at the transmembrane helix to Thr and demonstrated the association of the polymorphism with susceptibility to systemic lupus erythematosus (SLE) in Asians. In this study, we examined the impact of FCGR2B c.695T>C on the functional properties of FcgammaRIIB by expressing each allele product in a human B cell line ST486 lacking endogenous FcgammaRIIB. FcgammaRIIB 232Thr was found to be significantly less potent than wild-type 232Ile in inhibiting B cell receptor (BCR)-mediated phosphatidylinositol-3,4,5-trisphosphate accumulation, Akt and PLCgamma2 activation and calcium mobilization, and to display decreased levels of tyrosine phosphorylation and SH2-containing 5'-inositolphosphate phosphatase recruitment compared with 232Ile after IgG Fc-mediated coligation with BCR. Notably, a quantitative analysis of the subcellular distribution of FcgammaRIIB using 125I-labeled anti-FcgammaRIIB revealed that FcgammaRIIB 232Thr is less effectively distributed to detergent-insoluble lipid rafts than 232Ile, findings in accordance with the importance of the transmembrane amino acid residues, in particular large hydrophobic amino acids including Ile, in the association of membrane proteins with lipid rafts. Given the crucial roles of lipid rafts in integrating BCR signaling, decreased association of FcgammaRIIB 232Thr could contribute to its impaired inhibitory potential. Collectively, the present findings indicate that the Ile232Thr substitution affects the localization and function of FcgammaRIIB and that the molecular mechanism may link the polymorphism and susceptibility to SLE.

The Nipah Virus Fusion Protein Is Cleaved within the Endosomal Compartment

Nipah virus (NiV) is a recently emerged and highly pathogenic paramyxovirus that causes a systemic infection in animals and humans and can infect a wide range of cultured cells. Interestingly, the NiV fusion (F) protein has a single arginine at the cleavage site similar to paramyxoviruses that are activated by exogenous trypsin-like enzymes only present in specific cells and tissues and therefore only cause localized infections. We show here that NiV F activation is not mediated by an exogenous serum protease but by an endogenous ubiquitous cellular protease after endocytosis of the protein. In addition to endocytosis, acidification of the endosome is a prerequisite for F cleavage. These results show that activation of the NiV F protein depends on a type of proteolytic cleavage that is clearly different from what is known for other paramyxoviral and orthomyxoviral fusion proteins. To our knowledge, this is the first example of a viral class I fusion protein whose activation depends on clathrin-mediated constitutive endocytosis.

Tissue distribution of the “N-end rule” ubiquitin-conjugating enzyme, HR6, in the rat

The conjugation of multiple ubiquitin molecules is required for recognition and degradation of a protein by the proteasome. The ubiquitination pathway responsible for the bulk of constitutive protein degradation targets proteins carrying basic or large hydrophobic amino acids at the N-terminus. In mammalian cells, this "N-end rule" pathway requires the ubiquitin-conjugating enzyme HR6. Until now, it has not been known which mammalian tissues and cell types predominantly utilize this pathway for degradation. Therefore, the distribution and intracellular localization of HR6 was determined by indirect immunofluorescence techniques and protein blotting of adult rat tissues. Intense immunoreactivity against HR6 was detected in various epithelia, muscle, testis, peripheral neurons, chromaffin cells and macrophages, whereas lower HR6 protein levels were found in the gut or in the kidney. Autonomic and sensory neurons, glandular cells and spermatocytes revealed prominent nuclear HR6 immunoreactivity. Plasma membrane labeling was observed in peripheral neurons, spermatocytes and skeletal muscle cells. Smooth muscle cells, macrophages, endothelial and epithelial cells exhibited primarily cytoplasmic staining. The clear differences in the regional and intracellular distribution of HR6 are suggestive for the involvement of N-end rule protein degradation in various physiological processes dependent on cell type and subcellular structure.

Preliminary crystallographic characterization of BSAP, an extracellular aminopeptidase fromBacillus subtilis

The extracellular aminopeptidase from Bacillus subtilis (BSAP) has recently been cloned, overexpressed and purified from Escherichia coli. It is a monomer with a molecular weight of 46 425 Da, consisting of 425 amino-acid residues and a double-zinc catalytic centre. The recombinant enzyme was found to be stable for 20 min at 353 K, to function optimally in the pH range 8-9 and to prefer basic and large hydrophobic N-terminal amino acids in peptide and protein substrates. As such, this enzyme can be used as a representative model for structural, functional and mechanistic studies of monomeric double-zinc aminopeptidases, many of which have been found to be involved in medically important biological activities. In this report, the crystallization and preliminary crystallographic characterization of wild-type BSAP are described. Two different crystal forms are reported, of which the hexagonal form H2 is the more suitable for structural study, with average unit-cell dimensions a = b = 226.5, c = 42.8 A. A full diffraction data set has been collected from such a crystal of the native enzyme (2.2 A resolution, 91.2% completeness, R(merge) = 7.1%). A multiwavelength anomalous diffraction (MAD) data set was collected on native (zinc-containing) BSAP at three wavelengths around the zinc absorption edge (peak data set at 2.5 A resolution, 98.8% completeness, R(merge) = 5.3%). These diffraction data were collected at 95-100 K using a synchrotron X-ray source and a CCD area detector. The data are currently being used to obtain crystallographic phasing and to determine the detailed three-dimensional structure of the enzyme.

Biochemical characterization of prostasin, a channel activating protease

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1′, P2′, and P3′ displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4′ had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC α- and β-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1′ explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.

In Vitro Modification of Human Centromere Protein CENP-C Fragments by Small Ubiquitin-like Modifier (SUMO) Protein: DEFINITIVE IDENTIFICATION OF THE MODIFICATION SITES BY TANDEM MASS SPECTROMETRY ANALYSIS OF THE ISOPEPTIDES

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif psiKXE/D, where psi represents a large hydrophobic amino acid, and X represents any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.

Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini.

Fatty acid desaturases (FADs) play a prominent role in plant lipid metabolism and are located in various subcellular compartments, including the endoplasmic reticulum (ER). To investigate the biogenesis of ER-localized membrane-bound FADs, we characterized the mechanisms responsible for insertion of Arabidopsis FAD2 and Brassica FAD3 into ER membranes and determined the molecular signals that maintain their ER residency. Using in vitro transcription/translation reactions with ER-derived microsomes, we show that both FAD2 and FAD3 are efficiently integrated into membranes by a co-translational, translocon-mediated pathway. We also demonstrate that while the C-terminus of FAD3 (-KSKIN) contains a functional prototypic dilysine ER retrieval motif, FAD2 contains a novel C-terminal aromatic amino acid-containing sequence (-YNNKL) that is both necessary and sufficient for maintaining localization in the ER. Co-expression of a membrane-bound reporter protein containing the FAD2 C-terminus with a dominant-negative mutant of ADP-ribosylation factor (Arf)1 abolished transient localization of the reporter protein in the Golgi, indicating that the FAD2 peptide signal acts as an ER retrieval motif. Mutational analysis of the FAD2 ER retrieval signal revealed a sequence-specific motif consisting of Phi-X-X-K/R/D/E-Phi-COOH, where -Phi- are large hydrophobic amino acid residues. Interestingly, this aromatic motif was present in a variety of other known and putative ER membrane proteins, including cytochrome P450 and the peroxisomal biogenesis factor Pex10p. Taken together, these data describe the insertion and retrieval mechanisms of FADs and define a new ER localization signal in plants that is responsible for the retrieval of escaped membrane proteins back to the ER.

Analysis of residues determining specificity of Vibrio cholerae TonB1 for its receptors.

In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex. Vibrio cholerae encodes two TonB proteins, one of which, TonB1, recognizes only a subset of V. cholerae TonB-dependent receptors and does not facilitate transport through Escherichia coli receptors. To investigate the receptor specificity exhibited by V. cholerae TonB1, chimeras were created between V. cholerae TonB1 and E. coli TonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus of V. cholerae TonB1 were identified that allowed TonB1 to recognize E. coli receptors and at least one V. cholerae TonB2-dependent receptor. This indicates that the very C-terminal end of V. cholerae TonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching between V. cholerae and E. coli receptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box in E. coli receptors with an aromatic residue allowed these receptors to use V. cholerae TonB1. Further, site-directed mutagenesis of the TonB box -1 residue in a V. cholerae TonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition of V. cholerae TonB1. These data suggest that the TonB box -1 position controls productive interactions with V. cholerae TonB1.