scholarly journals C-Terminal Truncations of the Saccharomyces cerevisiae PMA1 H+-ATPase Have Major Impacts on Protein Conformation, Trafficking, Quality Control, and Function

2013 ◽  
Vol 13 (1) ◽  
pp. 43-52 ◽  
Author(s):  
A. Brett Mason ◽  
Kenneth E. Allen ◽  
Carolyn W. Slayman
Keyword(s):  
Amino Acids ◽  
Quality Control ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Amino Acid Residues ◽  
Membrane Expression ◽  
C Terminus ◽  
Conditional Expression ◽  
Membrane Spanning ◽  
And Function

ABSTRACTThe C-terminal tail of yeast plasma membrane (PM) H+-ATPase extends approximately 38 amino acids beyond the final membrane-spanning segment (TM10) of the protein and is known to be required for successful trafficking, stability, and regulation of enzyme activity. To carry out a detailed functional survey of the entire length of the tail, we generated 15 stepwise truncation mutants. Eleven of them, lacking up to 30 amino acids from the extreme terminus, were able to support cell growth, even though there were detectable changes in plasma membrane expression, protein stability, and ATPase activity. Three functionally distinct regions of the C terminus could be defined. (i) Truncations upstream of Lys889, removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala881(at the end of TM10) to Gly888is required for stable folding and PM targeting. (ii) The stretch between Lys889and Lys916, a region known to be subject to kinase-mediated posttranslational modification, was shown here to be ubiquitinated in carbon-starved cells as part of cellular quality control and to be essential for normal ATPase folding and stability, as well as for autoinhibition of ATPase activity during glucose starvation. (iii) Finally, removal of even one or two residues (Glu917and Thr918) from the extreme C terminus led to visibly reduced expression of the ATPase at the plasma membrane. Thus, the C terminus is much more than a simple appendage and profoundly influences the structure, biogenesis, and function of the yeast H+-ATPase.

scholarly journals Identification of Heterotrimeric G Protein γ3 Subunit in Rice Plasma Membrane

2018 ◽  
Vol 19 (11) ◽  
pp. 3591 ◽  
Author(s):  
Aki Nishiyama ◽  
Sakura Matsuta ◽  
Genki Chaya ◽  
Takafumi Itoh ◽  
Kotaro Miura ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Higher Plants ◽  
Rice Genome ◽  
Amino Acid Residues ◽  
Subunit Gene ◽  
Α Subunit ◽  
Γ Subunit ◽  
Domain Antibody ◽  
C Terminus ◽  
Liquid Chromatography Tandem Mass

Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical β subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and β subunits (Gβ), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.

scholarly journals The role of prolines and glycine in the transmembrane domain of LAT

2020 ◽  
Author(s):  
Daniela Glatzová ◽  
Harsha Mavila ◽  
Maria Chiara Saija ◽  
Tomáš Chum ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Surface Expression ◽  
Transmembrane Segment ◽  
Proline Residue ◽  
And Function ◽  
The Impact

ABSTRACTLAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

Structure and function of the NHE1 isoform of the Na+/H+ exchanger

2002 ◽  
Vol 80 (5) ◽  
pp. 499-508 ◽  
Author(s):  
Emily Slepkov ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Integral Membrane Protein ◽  
Amino Acid Residues ◽  
Transmembrane Helices ◽  
Cytoskeletal Organization ◽  
Transmembrane Segments ◽  
And Function ◽  
Extracellular Sodium

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cyto skel etal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.Key words: cation transport, intracellular pH, membrane proteins, Na+/H+ exchanger.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals AnkB, a Periplasmic Ankyrin-Like Protein inPseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide

2000 ◽  
Vol 182 (16) ◽  
pp. 4545-4556 ◽  
Author(s):  
Michael L. Howell ◽  
Eyad Alsabbagh ◽  
Ju-Fang Ma ◽  
Urs A. Ochsner ◽  
Martin G. Klotz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles ◽  
Gene Encoding ◽  
Rnase Protection ◽  
C Terminus ◽  
Hydrophobic Amino Acids ◽  
Increased Sensitivity ◽  
Membrane Spanning

ABSTRACT In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. TheankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location ofankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is ∼65% α-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katBand ankB are part of a small operon whose transcription is induced dramatically by H2O2, and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of anankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H2O2, phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H2O2 detoxification.

scholarly journals Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

PLoS ONE ◽  
2012 ◽  
Vol 7 (12) ◽  
pp. e52161 ◽  
Author(s):  
Sylvain M. Le Gall ◽  
Julie Legrand ◽  
Mohcine Benbijja ◽  
Hanaa Safya ◽  
Karim Benihoud ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
P2x7 Receptor ◽  
Double Negative ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
And Function

Hereditary Thrombocytosis: The Thrombopoietin Receptor c-MPL Can Activate Its Downstream Pathway without Full Glycosylation and Membrane Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2374-2374
Author(s):  
Clemens Stockklausner ◽  
Nicole Echner ◽  
Anne-Christine Klotter ◽  
Isabelle Nadine Kuhlee ◽  
Andreas E Kulozik
Keyword(s):  
Plasma Membrane ◽  
Ligand Binding ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Direct Consequence ◽  
Compound Heterozygous ◽  
Membrane Expression ◽  
Receptor Dimerization ◽  
Hereditary Thrombocytopenia ◽  
And Function

Abstract Abstract 2374 Thrombopoiesis is tightly regulated by the interaction between thrombopoietin (TPO) and its receptor c-Mpl. Receptor binding also leads to the clearance of TPO from the plasma thus establishing a negative feedback loop. However, it is still an open question how the receptor activates its downstream pathway. Alternative models posit that ligand binding either results in receptor dimerization in the plasma membrane or leads to conformational change of preformed receptor dimers. Several mutations in the TPO and the c-Mpl receptor genes have been linked to either hereditary thrombocytopenia or thrombocytosis. We focused on mutations in the extracellular part of the c-Mpl receptor, where ligand binding and receptor dimerization occur. Mutated homozygous c-Mpl R102P and compound heterozygous R102P/F104S receptors cause severe hereditary thrombocytopenia. In contrast, the homozygous c-Mpl P106L mutation was found in patients with hereditary thrombocytosis. We now addressed the question of how the disparate phenotype of mutations in the same domain of the c-Mpl receptor can be explained. We first functionally analyzed and compared normal with mutated R102P, F104S and P106L c-Mpl receptors in transfected HeLa and BA/F3 cells and found that the normal and the F104S c-Mpl receptors are glycosylated normally by the Golgi apparatus and reach the plasma membrane. In contrast, the R102P and P106L mutated receptors are not fully glycosylated, do not reach the plasma membrane and are atypically distributed in the ER. Functional analysis of the TPO/c-Mpl signaling pathway in BA/F3 cells showed decreased phosphorylation of Stat3, Stat5 and Erk1/2 with the R102P and F104S mutants when compared to normal. By contrast, TPO/c-Mpl signaling was up-regulated in cells transfected with the P106L-mutated receptor. Moreover, the P106L mutant, but not the other mutant receptors, enhanced ligand-independent growth of transfected BA/F3 cells. Despite of their opposite function, the TPO plasma levels of patients carrying both, homozygous R102P and P106L mutations were elevated 10 to 20-fold compared to normal and heterozygous individuals. This finding, together with their impaired glycosylation and inability to reach the plasma membrane, suggests that these mutants do not bind and internalize their ligand. TPO binding and degradation thus requires the receptor to be expressed at the plasma membrane, whereas, surprisingly, c-Mpl P106L activated its signaling pathway in a ligand independent fashion. Correct receptor processing and function can thus be separated. This indicates that TPO binding is required for regulation but that the constitutive activation of c-Mpl P106L is a likely direct consequence of premature receptor dimerization in the ER, auto-phosphorylation and subsequent activation of downstream targets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Plasma Membrane and Nuclear Localization of G Protein–coupled Receptor Kinase 6A

2007 ◽  
Vol 18 (8) ◽  
pp. 2960-2969 ◽  
Author(s):  
Xiaoshan Jiang ◽  
Jeffrey L. Benovic ◽  
Philip B. Wedegaertner
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
G Protein ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Amphipathic Helix ◽  
G Protein Coupled Receptor ◽  
C Terminus ◽  
Acidic Residues ◽  
G Protein Coupled

G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

scholarly journals Syntenin-1 Is a New Component of Tetraspanin-Enriched Microdomains: Mechanisms and Consequences of the Interaction of Syntenin-1 with CD63

2006 ◽  
Vol 26 (20) ◽  
pp. 7707-7718 ◽  
Author(s):  
Nadya Latysheva ◽  
Gairat Muratov ◽  
Sundaresan Rajesh ◽  
Matthew Padgett ◽  
Neil A. Hotchin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Pdz Domain ◽  
Binding Pocket ◽  
Resonance Spectroscopy ◽  
Nmr Studies ◽  
Transmembrane Receptors ◽  
C Terminus ◽  
Elevated Expression

ABSTRACT Tetraspanins are clustered in specific microdomains (named tetraspanin-enriched microdomains, or TERM) in the plasma membrane and regulate the functions of associated transmembrane receptors, including integrins and receptor tyrosine kinases. We have identified syntenin-1, a PDZ domain-containing protein, as a new component of TERM and show that syntenin-1 specifically interacts with the tetraspanin CD63. Detailed biochemical and heteronuclear magnetic resonance spectroscopy (NMR) studies have demonstrated that the interaction is mediated by the C-terminal cytoplasmic region of the tetraspanin and the PDZ domains of syntenin-1. Upon interaction, NMR chemical shift perturbations were predominantly localized to residues around the binding pocket of PDZ1, indicating a specific mode of recognition of the cytoplasmic tail of CD63. In addition, the C terminus of syntenin-1 has a stabilizing role in the CD63-syntenin-1 association, as deletion of the last 17 amino acids abolished the interaction. The CD63-syntenin-1 complex is abundant on the plasma membrane, and the elevated expression of the wild-type syntenin-1 slows down constitutive internalization of the tetraspanin. Furthermore, internalization of CD63 was completely blocked in cells expressing a syntenin-1 mutant lacking the first 100 amino acids. Previous results have shown that CD63 is internalized via AP-2-dependent mechanisms. Hence, our data indicate that syntenin-1 can counteract the AP-2-dependent internalization and identify this tandem PDZ protein as a new regulator of endocytosis.

scholarly journals C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity

2017 ◽  
Vol 28 (3) ◽  
pp. 452-462 ◽  
Author(s):  
Madhavan Chalat ◽  
Kody Moleschi ◽  
Robert S. Molday
Keyword(s):  
Endoplasmic Reticulum ◽  
Atpase Activity ◽  
Deletion Mutants ◽  
Serine Residue ◽  
Cellular Processes ◽  
Phospholipid Asymmetry ◽  
C Terminus ◽  
And Function ◽  
Phospholipid Flippase

ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2.

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