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

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.

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NMR Studies of the Reaction of Amino Acids with Aqueous Chlorine

Nuclear Magnetic Resonance Spectroscopy in Environment Chemistry ◽

10.1093/oso/9780195097511.003.0013 ◽

1997 ◽

Author(s):

Frank E. Scully, Jr ◽

Barbara Conyers

Keyword(s):

Amino Acids ◽

Aquatic Organisms ◽

Nondestructive Method ◽

Resonance Spectroscopy ◽

Nmr Studies ◽

Wastewater Disinfection ◽

Water And Wastewater ◽

Gas Chromatography Mass Spectroscopy ◽

The Relationship ◽

By Products

Over the past 20 years, gas chromatography/mass spectroscopy (GC/MS) has been widely used to identify trace organic environmental contaminants and to study the mechanisms of the formation or transformation of organic compounds either by natural or man-made processes. In the area of water and wastewater disinfection, GC/MS has been highly successful in identifying numerous volatile organic chlorination by-products, some of which may pose undesirable health risks to humans and aquatic organisms at concentrations found in some waters. However, despite a considerable amount of research in this area much of the chemistry continues to be poorly understood. Analysis of trace organics by GC/MS relies on the assumption that the compounds to be analyzed are (1) volatile and (2) thermally stable to GC temperatures as high as 300 °C. Because nuclear magnetic resonance spectroscopy (NMR) is a mild and nondestructive method of analysis, it can reveal reactions that occur in water that cannot be observed by GC/MS. Until recently the reactions of amino acids with two or more equivalents of aqueous chlorine were believed to produce aldehydes and nitriles according to equation (1). LeCloirec and Martin have reported that the formation of nitriles in such situations may come in part from the reaction of monochloramine with aldehydes (equation (2)). Because reaction (2) may affect the distribution of products in reaction (1), it was important to determine the relationship between these two reactions. This chapter will review the applications of NMR we have used in studies of the products formed upon chlorination of amino acids.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e07-01-0013 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2960-2969 ◽

Cited By ~ 32

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.

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Positive and Negative Regulation of Poly(A) Nuclease

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5521-5533.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5521-5533 ◽

Cited By ~ 57

Author(s):

David A. Mangus ◽

Matthew C. Evans ◽

Nathan S. Agrin ◽

Mandy Smith ◽

Preetam Gongidi ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Binding Pocket ◽

Negative Regulation ◽

Single Amino Acid ◽

Carboxy Terminus ◽

Protein Protein Interactions ◽

C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

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Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor

Biochemical Journal ◽

10.1042/bj3260497 ◽

1997 ◽

Vol 326 (2) ◽

pp. 497-505 ◽

Cited By ~ 29

Author(s):

Kristin DENZER ◽

Birgit WEBER ◽

Annette HILLE-REHFELD ◽

Kurt VON FIGURA ◽

Regina POHLMANN

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Cytoplasmic Tail ◽

Plasma Membrane Protein ◽

The Third ◽

C Terminus ◽

Critical Residue ◽

Internalization Rate ◽

Mannose 6 Phosphate ◽

Mpr 46

The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant.

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Sorting Signals within the Saccharomyces cerevisiae Sporulation-Specific Dityrosine Transporter, Dtr1p, C Terminus Promote Golgi-to-Prospore Membrane Transport

Eukaryotic Cell ◽

10.1128/ec.00151-08 ◽

2008 ◽

Vol 7 (10) ◽

pp. 1674-1684 ◽

Cited By ~ 7

Author(s):

Masayo Morishita ◽

JoAnne Engebrecht

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Plasma Membrane ◽

Membrane Transport ◽

Transmembrane Domain ◽

Spore Wall ◽

Bilayer Membrane ◽

C Terminus ◽

Prospore Membrane ◽

Dividing Cells

ABSTRACT During sporulation in Saccharomyces cerevisiae, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a cis-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a trans-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.

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Cooperative binding of the tandem WW domains of PLEKHA7 to PDZD11 promotes conformation-dependent interaction with tetraspanin 33

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012987 ◽

2020 ◽

Vol 295 (28) ◽

pp. 9299-9312 ◽

Cited By ~ 1

Author(s):

Florian Rouaud ◽

Francesca Tessaro ◽

Laura Aimaretti ◽

Leonardo Scapozza ◽

Sandra Citi

Keyword(s):

Ternary Complex ◽

Hot Spot ◽

Pdz Domain ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Pleckstrin Homology Domain ◽

Ww Domains ◽

C Terminus ◽

Toxin Receptor ◽

Accessible Surface

Pleckstrin homology domain–containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain–containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin–mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S-transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a “hydrophobic hot spot” region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7–PDZD11 interaction modulate the ligand-binding properties of PLEKHA7.

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

Eukaryotic Cell ◽

10.1128/ec.00201-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 43-52 ◽

Cited By ~ 14

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.

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C-Terminal Signals Regulate Targeting of Glycosylphosphatidylinositol-Anchored Proteins to the Cell Wall or Plasma Membrane in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00148-08 ◽

2008 ◽

Vol 7 (11) ◽

pp. 1906-1915 ◽

Cited By ~ 18

Author(s):

Yuxin Mao ◽

Zimei Zhang ◽

Charles Gast ◽

Brian Wong

Keyword(s):

Amino Acids ◽

Cell Wall ◽

Candida Albicans ◽

Plasma Membrane ◽

Fluorescent Protein ◽

Fluorescent Reporters ◽

C Terminus ◽

Peptide Signals ◽

Gpi Proteins ◽

Green Fluorescent

ABSTRACT Fungal glycosylphosphatidylinositol (GPI)-anchored proteins localize to the plasma membrane (PM), cell wall (CW), or both. To study signals that regulate PM versus CW targeting in Candida albicans, we (i) fused the N and/or C termini of the GPI CW protein Hwp1p and the GPI PM protein Ecm331p to green fluorescent protein (GFP) and (ii) expressed and localized the resulting fusions. Forty-seven amino acids from the C terminus of Hwp1p were sufficient to target GFP to the CW, and 66 amino acids from the C terminus of Ecm331p were sufficient to target GFP to the PM. Truncation and mutagenesis studies showed that G390 was the ω cleavage site in Ecm331p. Domain exchange and mutagenesis studies showed that (i) the 5 amino acids immediately N-terminal to the ω sites (the ω − 5 to ω − 1 amino acids) played key roles in targeting to the PM or CW; (ii) KK and FE residues at positions ω − 1 and ω − 2, respectively, targeted to the PM and CW; and (iii) a loss of I at position ω − 5 increased PM retention. Small fluorescent reporters can be used to study the peptide signals that regulate PM versus CW targeting of GPI proteins and may be useful for identifying proteins that interact with key targeting signals.

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Plasma membrane Ca2+-ATPase associates with CLP36, α-actinin and actin in human platelets

Thrombosis and Haemostasis ◽

10.1160/th06-08-0438 ◽

2007 ◽

Vol 97 (04) ◽

pp. 587-597 ◽

Cited By ~ 23

Author(s):

Larry Bozulic ◽

Mohammad Malik ◽

David Powell ◽

Adrian Nanez ◽

Andrew Link ◽

...

Keyword(s):

Plasma Membrane ◽

Pdz Domain ◽

Gel Filtration ◽

Human Platelets ◽

Cytoskeletal Proteins ◽

Pdz Domains ◽

Glutathione S Transferase ◽

Spectrometry Analysis ◽

C Terminus ◽

Domain Interactions

SummaryThe plasma membrane Ca2+-ATPase (PMCA) plays an essentialrole in maintaining low cytosolic Ca2+ in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts viaits C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the lastten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pumpto the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitationassays coupled with liquid chromatography/tandemmass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing proteinassociated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extractusing a fusion protein containing the C-terminal PDZ domainbinding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized plateletsdemonstrated the existence of a 1,000-kDa complex containingPMCA and CLP36, and in addition, α -actinin and actin. Immunoflourescencemicroscopy confirmed the co-localization ofPMCA with CLP36 in resting and activated platelets. Taken togetherthese results suggest that PMCA is localized in non-filamentousactin complexes in resting platelets by means of PDZdomain interactions and then associates with the actin cytoskeletonduring cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine andtyrosine phosphorylation events previously described in humanplatelets, PMCA function may be regulated by interactions withanchoring and cytoskeletal proteins.

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Interaction of G-protein-coupled receptors with synaptic scaffolding proteins

Biochemical Society Transactions ◽

10.1042/bst0300464 ◽

2002 ◽

Vol 30 (4) ◽

pp. 464-468 ◽

Cited By ~ 20

Author(s):

H.-J. Kreienkamp ◽

M. Soltau ◽

D. Richter ◽

T. Böckers

Keyword(s):

Rat Brain ◽

Somatostatin Receptor ◽

Pdz Domain ◽

Interacting Protein ◽

Transmembrane Receptors ◽

C Terminus ◽

Large N ◽

Brain Extracts ◽

The Central Nervous System

The calcium-independent receptors for latrotoxin (CIRL1-CIRL3) constitute a family of seven-transmembrane receptors with an unsually large N-terminal extracellular domain which comprises several motifs usually found in cell adhesion molecules. By yeast two-hybrid screening, we have identified the intracellular C-termini of CIRL1 and CIRL2 as interaction partners of the PDZ domain of the proline-rich synapse-associated protein (ProSAP)/somatostatin receptor-interacting protein (SSTRIP) family of postsynaptic proteins (SSTRIP, ProSAP1 and ProSAP2, also known as shank1-shank3 respectively). Overlay assays indicate that the ProSAP1/shank2 PDZ domain in particular interacts strongly with the C-terminus of CIRL1 and CIRL2. Co-immuno-precipitation of ProSAP1 and CIRL1 (but not CIRL2) from rat brain extracts indicates that this interaction also occurs in vivo in rat brain. The known postsynaptic localization of ProSAP1, as well as our observation that CIRL1 (but not CIRL2) is enriched in postsynaptic density preparations from the rat brain, suggests that CIRL1 is localized pre- as well as post-synaptically in the central nervous system.

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