Targeting and retention of type 1 ryanodine receptors to the endoplasmic reticulum

Most ryanodine receptors and their relatives, inositol 1,4,5-trisphosphate receptors, are expressed in the sarcoplasmic or endoplasmic reticulum (ER), where they mediate Ca(2+) release. We expressed fragments of ryanodine receptor type 1 (RyR1) in COS cells alone or fused to intercellular adhesion molecule-1 (ICAM-1), each tagged with yellow fluorescent protein, and used confocal imaging and glycoprotein analysis to identify the determinants of ER targeting and retention. Single transmembrane domains (TMD) of RyR1 taken from the first (TMD1-TMD2) or last (TMD5-TMD6) pair were expressed in the ER membrane. TMD3-TMD4 was expressed in the outer mitochondrial membrane. The TMD outer pairs (TMD1-TMD2 and TMD5-TMD6) retained ICAM-1, a plasma membrane-targeted protein, within the ER membrane. TMD1 alone provided a strong ER retention signal and TMD6 a weaker signal, but the other single TMD were unable to retain ICAM-1 in the ER. We conclude that TMD1 provides the first and sufficient signal for ER targeting of RyR1. The TMD outer pairs include redundant ER retention signals, with TMD1 providing the strongest signal.     

Mutational analysis of the Aspergillus ambient pH receptor PalH underscores its potential as a target for antifungal compounds

The pal/RIM ambient pH signalling pathway is crucial for the ability of pathogenic fungi to infect hosts. The Aspergillus nidulans 7‐TMD receptor PalH senses alkaline pH, subsequently facilitating ubiquitination of the arrestin PalF. Ubiquitinated PalF triggers downstream signalling events. The mechanism(s) by which PalH transduces the alkaline pH signal to PalF is poorly understood. We show that PalH is phosphorylated in a signal dependent manner, resembling mammalian GPCRs, although PalH phosphorylation, in contrast to mammalian GPCRs, is arrestin dependent. A genetic screen revealed that an ambient‐exposed region comprising the extracellular loop connecting TM4‐TM5 and ambient‐proximal residues within TM5 is required for signalling. In contrast, substitution by alanines of four aromatic residues within TM6 and TM7 results in a weak ‘constitutive’ activation of the pathway. Our data support the hypothesis that PalH mechanistically resembles mammalian GPCRs that signal via arrestins, such that the relative positions of individual helices within the heptahelical bundle determines the Pro316‐dependent transition between inactive and active PalH conformations, governed by an ambient‐exposed region including critical Tyr259 that potentially represents an agonist binding site. These findings open the possibility of screening for agonist compounds stabilizing the inactive conformation of PalH, which might act as antifungal drugs against ascomycetes.     

Receptor-independent Ambient pH Signaling by Ubiquitin Attachment to Fungal Arrestin-like PalF

The seven-transmembrane receptor PalH and its coupled, positive-acting arrestin-like protein PalF are key components of a molecular sensor that in Aspergillus nidulans and other ascomycete fungi mediates activation of an intracellular signaling cascade by alkaline ambient pH. PalF is ubiquitinated in an alkaline pH- and PalH-dependent manner. We show here that PalF assists the plasma membrane localization of PalH and that PalF overexpression slightly hypersensitizes the pathway to alkaline pH but does not bypass the need for the ambient pH signal receptor in signaling. In contrast, covalent attachment of Ub to PalF activates the signaling pathway under acidic pH conditions in which the pathway is normally inactive, demonstrating a positive role for ubiquitination. We further show that PalF acts upstream of, or in concert with, the Bro1 domain-containing pH signaling protein PalC, which is normally recruited to cortical structures likely to represent active pH signaling foci under neutral/alkaline pH conditions. In agreement with its pathway-activating consequences, expression of PalF-Ub also promotes PalC cortical recruitment under acidic conditions. Notably, our data establish that expression of PalF-Ub, at approximately physiological levels, in a null palH background leads to a considerable degree of signaling even in the complete absence of the receptor. Thus PalF ubiquitination is a key, perhaps the sole, molecular trigger required for transmitting the alkaline pH signal to the downstream elements of the pathway.     

Structure and metal ion binding of the first transmembrane domain of DMT1

DMT1 is an integral membrane protein with 12 putative transmembrane domains. As a divalent metal ion transporter, it plays an important role in metal ion homeostasis from bacteria to human. Loss-function mutations at the conserved motif DPGN located within the first transmembrane domain (TMD1) of DMT1 indicate the significance of TMD1 in the biological function of the protein. In the present work, we study the structure, topology and metal ion binding of DMT1-TMD1 peptide by nuclear magnetic resonance using sodium dodecyl sulfate and dodecylphosphocholine micelles as membrane mimics. We find that the peptide forms an α-helix-extended segment-α-helix configuration in which the motif DPGN locates at the central flexible region. The N-terminal part of the peptide is deeply embedded in micelles, while the motif section and the C-terminal part are close to the surface of micelles. The peptide can bind to Mn2+ and Co2+ ions by the side chains of the negatively charged residues in the motif section and the C-terminal part of TMD1. The crucial role of the central flexible region and the C-terminal part of TMD1 in metal ion capture is confirmed by the binding of the N-terminal part truncated TMD1 to metal ions.     

Functional role of the conserved proline in helix 6 of the human bradykinin B2 receptor

Pro258 in transmembrane domain (TMD) 6 of the bradykinin (BK) B₂ receptor (B₂R) is highly conserved among G-protein coupled receptors (GPCRs). Using mutagenesis, we show that Pro258 is required for normal trafficking of the receptor to the plasma membrane and that mutation of Pro258 to Ala or Leu but not Gly, enhances BK efficacy to induce receptor activation. Furthermore, P258A mutation suppresses the constitutive activity of a constitutively activated N113A-B₂R mutant but preserves the antagonist to agonist efficacy shift previously observed on the N113A single mutant. Our data suggest that Pro258 in TMD6 is required for agonist-independent activation of the B₂R and that straightening of TMD6 at the Pro-kink might favor G-protein coupling. It is also shown that Asn113 is a contact point of BK interaction and it is proposed that the release of a TMD3-TMD6 interaction involving Asn113 is crucial for the efficacy shift from antagonism toward agonism.     

Mutational Analysis of the Amino Acid Residues Essential for the cis and trans Cleavage Activity of the Potato Virus Y 50-kDa Protease

To examine, the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234→Tyr, Asp269→Asn, or Cys339→Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339→Ser replacement retained a reduced proteolytic activity.     

A conserved cationic motif enhances membrane binding and insertion of the chloride intracellular channel protein 1 transmembrane domain

The chloride intracellular channel protein 1 (CLIC1) is unique among eukaryotic ion channels in that it can exist as either a soluble monomer or an integral membrane channel. CLIC1 contains no known membrane-targeting signal sequences and the environmental factors which promote membrane binding of the transmembrane domain (TMD) are poorly understood. Here we report a positively charged motif at the C-terminus of the TMD and show that it enhances membrane partitioning and insertion. A 30-mer TMD peptide was synthesized in which the positively charged motif was replaced by three glutamate residues. The peptide was examined in 2,2,2-trifluoroethanol (TFE), sodium dodecyl sulfate micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using size-exclusion chromatography, far-UV CD, and fluorescence spectroscopy. The motif appears to enhance membrane interaction via electrostatic contacts and functions as an electrostatic plug to anchor the TMD in membranes. In addition, the motif is also involved in orientating the TMD with respect to the cis and trans faces of the membrane. These findings shed light on the intrinsic and environmental factors that promote the spontaneous conversion of CLIC1 from a water-soluble to a membrane-bound protein.     

A motif rich in charged residues determines product specificity in isomaltulose synthase

Isomaltulose synthase (PalI) catalyzes hydrolysis of sucrose and formation of alpha-1,6 and alpha-1,1 bonds to produce isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofranose) and small amount of trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofranose). A potential isomaltulose synthase-specific motif ((325)RLDRD(329)), that contains a 'DxD' motif conserved in many glycosyltransferases, was identified based on sequence comparison with reference to the secondary structural features of PalI and homologs. Site-directed mutagenesis analysis of the motif showed that the four charged amino acid residues (Arg(325), Arg(328), Asp(327) and Asp(329)) influence the enzyme kinetics and determine the product specificity. Mutation of these four residues increased trehalulose formation by 17-61% and decreased isomaltulose by 26-67%. We conclude that the 'RLDRD' motif controls the product specificity of PalI.     

Enzymatic 7β hydroxylation of 4,4,10β-trimethyl-trans-decal-3β-ol (TMD)

4,4,10β-Trimethyl-trans-decal-3β-ol (TMD, 1), an inhibitor of 2,3-oxidosqualene cyclase, is converted by S₁₀ rat liver homogenate (RLH) to 4,4,10β-trimethyl-trans-decalin-3β,7β-diol (2) as its chief metabolite in 15 to 32% yield. Identification of 2 was accomplished by (a) gas-liquid chromatography/mass spectroscopy (GLC-MS) molecular-weight determination; (b) identity of GLC retention times with those of synthetic 2 and derived dione 15 on three columns; and (c) isotopic dilution and recrystallization to constant specific activity of both ³H-2 and its diacetate. Resolution of TMD was effected via formation of diastereomeric esters 18 and 19; and both d-TMD and l-TMD were found to be metabolized in RLH to 2, with d-TMD, the enantiomer having the nonsteroidal absolute configuration, being about twice as effectively hydroxylated.     

Insight into the structures of the second and fifth transmembrane domains of Slc11a1 in membrane mimics

Slc11a1 is an integral membrane protein with 12 putative transmembrane domains and functions as a pH‐coupled divalent metal cation transporter. In the present study, the structures of the peptides corresponding to the second and fifth transmembrane domains of Slc11a1 (from 88 to 109 for TMD2 and from 190 to 215 for TMD5) were determined in membrane‐mimic environments by CD and NMR techniques. It was demonstrated that TMD2 and TMD5 form an α‐helical structure in 30% 2,2,2‐trifluoroethanol (TFE) and 40% hexafluoro‐2‐propanol (HFIP) aqueous solution, respectively. The α‐helix of TMD5 displays a less space‐occupied face consisting of the residues Ala194, Gly197, Thr201, Ala204 and Gly208. The α‐helix is partially unfolded in the N‐terminal region when Gly197 is substituted by Val. The unfolding of the helix in the N‐terminal part and/or increase in volume at the less space‐occupied face of the helix may exert an effect on the arrangement of TMD5 in membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.