scholarly journals Calcium-dependent Plasma Membrane Binding and Cell Lysis by Perforin Are Mediated through Its C2 Domain

2004 ◽  
Vol 280 (9) ◽  
pp. 8426-8434 ◽  
Author(s):  
Ilia Voskoboinik ◽  
Marie-Claude Thia ◽  
Jamie Fletcher ◽  
Annette Ciccone ◽  
Kylie Browne ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Binding ◽  
Cell Lysis ◽  
C2 Domain ◽  
Calcium Dependent ◽  
Plasma Membrane Binding

scholarly journals gp96 Is a Human Colonocyte Plasma Membrane Binding Protein for Clostridium difficile Toxin A

2008 ◽  
Vol 76 (7) ◽  
pp. 2862-2871 ◽  
Author(s):  
Xi Na ◽  
Ho Kim ◽  
Mary P. Moyer ◽  
Charalabos Pothoulakis ◽  
J. Thomas LaMont
Keyword(s):  
Plasma Membrane ◽  
Clostridium Difficile ◽  
Binding Protein ◽  
Membrane Binding ◽  
Cell Surface Receptor ◽  
Toxin A ◽  
Surface Receptor ◽  
A Cell ◽  
Plasma Membrane Binding

ABSTRACT Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity.

scholarly journals Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 Domain

2006 ◽  
Vol 17 (1) ◽  
pp. 56-66 ◽  
Author(s):  
John H. Evans ◽  
Diana Murray ◽  
Christina C. Leslie ◽  
Joseph J. Falke
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Membrane Binding ◽  
Membrane Lipid ◽  
C2 Domain ◽  
Plasma Membrane Lipid ◽  
Protein Kinase Cα ◽  
Golgi Network

The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca2+-binding loops (CBLs) and PIP2 binding site (β-strands 3–4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP2.

scholarly journals HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

2020 ◽  
Vol 295 (52) ◽  
pp. 17950-17972
Author(s):  
Birgit Meusser ◽  
Bettina Purfuerst ◽  
Friedrich C. Luft
Keyword(s):  
Plasma Membrane ◽  
Mutational Analysis ◽  
Genetic Manipulation ◽  
Membrane Binding ◽  
Hek293 Cells ◽  
Binding Studies ◽  
Site Mutation ◽  
Terminal Protein ◽  
Hiv 1 ◽  
Plasma Membrane Binding

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

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