scholarly journals The Dense-Core Vesicle Maturation Protein CCCP-1 Binds RAB-2 and Membranes through its C-terminal Domain

2017 ◽  
Author(s):  
Jérôme Cattin-Ortolá ◽  
Irini Topalidou ◽  
Annie Dosey ◽  
Alexey J. Merz ◽  
Michael Ailion
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Dense Core ◽  
Binding Motif ◽  
Dense Core Vesicle ◽  
Terminal Domain ◽  
C Terminus

AbstractDense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.Synopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

Structure/function analysis of the C3a-receptor C-terminus

2000 ◽  
Vol 49 (1-2) ◽  
pp. 89
Author(s):  
B. Settmacher ◽  
C. Rheinheimer ◽  
M. Oppermann ◽  
H. Hamacher ◽  
D. Bock ◽  
...  
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
C Terminus

scholarly journals Structure-Function Analysis of the TibA Self-Associating Autotransporter Reveals a Modular Organization

2011 ◽  
Vol 79 (5) ◽  
pp. 1826-1832 ◽  
Author(s):  
Jean-Philippe Côté ◽  
Michael Mourez
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Extracellular Domain ◽  
Site Directed Mutagenesis ◽  
Modular Organization ◽  
Adhesion Properties ◽  
Content Type ◽  
C Terminus ◽  
Function Relationship ◽  
Insertion Mutants

ABSTRACTSome enterotoxigenicEscherichia colistrains express the TibA adhesin/invasin, a multifunctional autotransporter that mediates the autoaggregation of bacteria, biofilm formation, adhesion to cultured epithelial cells, and invasion of these cells. To elucidate the structure-function relationship in TibA, we generated mutants by transposon-based linker scanning mutagenesis and by site-directed mutagenesis. Several insertion mutants had a defect in either adhesion or autoaggregation. Mutants with a defect in autoaggregation were found in the N-terminal half of the extracellular domain, while mutants with a defect in adhesion were found in the C-terminal half. The deletion of the putative N-terminal autoaggregation domain abolished the autoaggregation of the bacteria but did not affect adhesion. The deletion of a proline-rich region located at the C terminus of the extracellular domain abolished the adhesion properties of TibA but did not affect invasion. This finding suggests that adhesion and invasion may rely on distinct mechanisms. Thus, our results reveal that TibA possesses a modular organization, with the extracellular domain being separated into an autoaggregation module and an adhesion module.

scholarly journals Protein Kinase C Phosphorylation of a γ-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization

2015 ◽  
Vol 290 (34) ◽  
pp. 20674-20686 ◽  
Author(s):  
Austin B. Keeler ◽  
Dietmar Schreiner ◽  
Joshua A. Weiner
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cortical Neurons ◽  
Lipid Binding ◽  
Binding Motif ◽  
Wild Type ◽  
Western Blots ◽  
Phospholipid Binding ◽  
C Terminus

The γ-protocadherins (γ-Pcdhs) are a family of 22 adhesion molecules with multiple critical developmental functions, including the proper formation of dendritic arbors by forebrain neurons. The γ-Pcdhs bind to and inhibit focal adhesion kinase (FAK) via a constant C-terminal cytoplasmic domain shared by all 22 proteins. In cortical neurons lacking the γ-Pcdhs, aberrantly high activity of FAK and of PKC disrupts dendrite arborization. Little is known, however, about how γ-Pcdh function is regulated by other factors. Here we show that PKC phosphorylates a serine residue situated within a phospholipid binding motif at the shared γ-Pcdh C terminus. Western blots using a novel phospho-specific antibody against this site suggest that a portion of γ-Pcdh proteins is phosphorylated in the cortex in vivo. We find that PKC phosphorylation disrupts both phospholipid binding and the γ-Pcdh inhibition of (but not binding to) FAK. Introduction of a non-phosphorylatable (S922A) γ-Pcdh construct into wild-type cortical neurons significantly increases dendrite arborization. This same S922A construct can also rescue dendrite arborization defects in γ-Pcdh null neurons cell autonomously. Consistent with these data, introduction of a phosphomimetic (S/D) γ-Pcdh construct or treatment with a PKC activator reduces dendrite arborization in wild-type cortical neurons. Together, these data identify a novel mechanism through which γ-Pcdh control of a signaling pathway important for dendrite arborization is regulated.

scholarly journals BAR Domain-Containing FAM92 Proteins Interact with Chibby1 To Facilitate Ciliogenesis

2016 ◽  
Vol 36 (21) ◽  
pp. 2668-2680 ◽  
Author(s):  
Feng-Qian Li ◽  
Xingwang Chen ◽  
Cody Fisher ◽  
Saul S. Siller ◽  
Klara Zelikman ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Cultured Cells ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Basal Bodies ◽  
Membrane Remodeling ◽  
Bar Domains

Chibby1 (Cby1) is a small, conserved coiled-coil protein that localizes to centrioles/basal bodies and plays a crucial role in the formation and function of cilia. During early stages of ciliogenesis, Cby1 is required for the efficient recruitment of small vesicles at the distal end of centrioles to facilitate basal body docking to the plasma membrane. Here, we identified family with sequence similarity 92, member A (FAM92A) and FAM92B, which harbor predicted lipid-binding BAR domains, as novel Cby1-interacting partners using tandem affinity purification and mass spectrometry. We found that in cultured cell lines, FAM92A colocalizes with Cby1 at the centrioles/basal bodies of primary cilia, while FAM92B is undetectable. In airway multiciliated cells, both FAM92A and -92B colocalize with Cby1 at the base of cilia. Notably, the centriolar localization of FAM92A and -92B depends largely on Cby1. Knockdown of FAM92A in RPE1 cells impairs ciliogenesis. Consistent with the membrane-remodeling properties of BAR domains, FAM92A and -92B in cooperation with Cby1 induce deformed membrane-like structures containing the small GTPase Rab8 in cultured cells. Our results therefore suggest that FAM92 proteins interact with Cby1 to promote ciliogenesis via regulation of membrane-remodeling processes.

Structure-function analysis of Ia molecules: in-phase insertion mutagenesis of the amino-terminal domain of the Eβk polypeptide chain

Biochimie ◽  
1988 ◽  
Vol 70 (7) ◽  
pp. 927-935
Author(s):  
Najet Rebaï ◽  
François Letourneur ◽  
Nilabh Shastri ◽  
Sylvie Marchetto ◽  
Michel Pierres ◽  
...  
Keyword(s):  
Structure Function ◽  
Polypeptide Chain ◽  
Function Analysis ◽  
Insertion Mutagenesis ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

scholarly journals gpwac of the T4-Type Bacteriophages: Structure, Function, and Evolution of a Segmented Coiled-Coil Protein That Controls Viral Infectivity

2005 ◽  
Vol 187 (3) ◽  
pp. 1055-1066 ◽  
Author(s):  
A. Letarov ◽  
X. Manival ◽  
C. Desplats ◽  
H. M. Krisch
Keyword(s):  
Bacteriophage T4 ◽  
Coiled Coil ◽  
Unknown Origin ◽  
Direct Consequence ◽  
Protein Protein Interaction ◽  
Terminal Domain ◽  
C Terminus ◽  
Coil Structure ◽  
Type Phage ◽  
Gene Orthologs

ABSTRACT The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the ∼50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.

scholarly journals Rab2 directs a stop-loss program

2009 ◽  
Vol 186 (6) ◽  
pp. 769-769
Author(s):  
Ben Short
Keyword(s):  
Small Gtpase ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Stop Loss

Small GTPase supports dense core vesicle maturation in worm neurons.

scholarly journals The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer

1999 ◽  
Vol 338 (2) ◽  
pp. 539-543 ◽  
Author(s):  
Judy CALLAGHAN ◽  
Anne SIMONSEN ◽  
Jean-Michel GAULLIER ◽  
Ban-Hock TOH ◽  
Harald STENMARK
Keyword(s):  
Protein Interactions ◽  
Quaternary Structure ◽  
Gradient Centrifugation ◽  
Active Form ◽  
Coiled Coil ◽  
Membrane Lipid ◽  
Small Gtpase ◽  
C Terminus ◽  
Early Endosomes ◽  
Heptad Repeats

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein–protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a ∼ 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the ∼ 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.

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