14-3-3 protein and ATRAP bind to the soluble class IIB phosphatidylinositol transfer protein RdgBβ at distinct sites

2012 ◽  
Vol 40 (2) ◽  
pp. 451-456 ◽  
Author(s):  
Shamshad Cockcroft ◽  
Kathryn Garner
Keyword(s):  
Membrane Trafficking ◽  
Biochemical Properties ◽  
Ang Ii ◽  
Lipid Transfer ◽  
Receptor Associated Protein ◽  
C Terminus ◽  
Phosphatidylinositol Transfer ◽  
Class Iib ◽  
Soluble Class ◽  
Multicellular Organisms

PITPs (phosphatidylinositol transfer proteins) are characterized by the presence of the PITP domain whose biochemical properties of binding and transferring PI (phosphatidylinositol) are well studied. Despite their wide-spread expression in both unicellular and multicellular organisms, they remain functionally uncharacterized. An emerging theme is that individual PITPs play highly specific roles in either membrane trafficking or signal transduction. To identify specific roles for PITPs, identification of interacting molecules would shed light on their molecular function. In the present paper, we describe binding partners for the class IIB PITP RdgBβ (retinal degeneration type Bβ). RdgBβ is a soluble PITP but is unique in that it contains a region of disorder at its C-terminus following its defining N-terminal PITP domain. The C-terminus of RdgBβ is phosphorylated at two serine residues, Ser274 and Ser299, which form a docking site for 14-3-3 proteins. Binding to 14-3-3 proteins protects RdgBβ from degradation that occurs at the proteasome after ubiquitination. In addition to binding 14-3-3, the PITP domain of RdgBβ interacts with the Ang II (angiotensin II)-associated protein ATRAP (Ang II receptor-associated protein). ATRAP is also an interacting partner for the AT1R (Ang II type 1 receptor). We present a model whereby RdgBβ functions by being recruited to the membrane by ATRAP and release of 14-3-3 from the C-terminus allows the disordered region to bind a second membrane to create a membrane bridge for lipid transfer, possibly under the control of Ang II.

The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein)

2011 ◽  
Vol 439 (1) ◽  
pp. 97-111 ◽  
Author(s):  
Kathryn Garner ◽  
Michelle Li ◽  
Natalie Ugwuanya ◽  
Shamshad Cockcroft
Keyword(s):  
Angiotensin Ii ◽  
Membrane Protein ◽  
Half Life ◽  
Integral Membrane Protein ◽  
Lipid Binding ◽  
Type I ◽  
Receptor Associated Protein ◽  
C Terminus ◽  
Phosphatidylinositol Transfer

PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser274 and Ser299) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.

Phosphatidylinositol-transfer protein and its homologues in yeast

2006 ◽  
Vol 34 (3) ◽  
pp. 377-380 ◽  
Author(s):  
P. Griac ◽  
R. Holic ◽  
D. Tahotna
Keyword(s):  
Membrane Trafficking ◽  
Diverse Group ◽  
Secretory Function ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylcholine Transfer Protein

Yeast Sec14p acts as a phosphatidylinositol/phosphatidylcholine-transfer protein in vitro. In vivo, it is essential in promoting Golgi secretory function. Products of five genes named SFH1–SFH5 (Sec Fourteen Homologues 1–5) exhibit significant sequence homology to Sec14p and together they form the Sec14p family of lipid-transfer proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism and membrane trafficking.

scholarly journals Deletion of 24 amino acids from the C-terminus of phosphatidylinositol transfer protein causes loss of phospholipase C-mediated inositol lipid signalling

1997 ◽  
Vol 324 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Simon PROSSER ◽  
Robert SARRA ◽  
Philip SWIGART ◽  
Andrew BALL ◽  
Shamshad COCKCROFT
Keyword(s):  
Amino Acids ◽  
Phospholipase C ◽  
Size Exclusion ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
C Terminus ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer protein α (PITPα) is a 32 kDa protein of 270 amino acids that is essential for phospholipase C-mediated phosphatidylinositol bisphosphate hydrolysis. In addition, it binds and transfers phosphatidylinositol and phosphatidylcholine between membrane compartments in vitro. Here we have used limited proteolysis of PITPα by subtilisin to identify the structural requirements for function. Digestion by subtilisin results in the generation of a number of slightly smaller peptide fragments, the major fragment being identified as a 29 kDa protein. The fragments were resolved by size-exclusion chromatography and were found to be totally inactive in both in vivo PLC reconstitution assays and in vitro phosphatidylinositol transfer assays. N-terminal sequencing and MS of the major 29 kDa fragment shows that cleavage occurs at the C-terminus of PITP at Met246, leading to a deletion of 24 amino acid residues. We conclude that the C-terminus plays an important role in mediating PLC signalling in vivo and lipid transfer in vitro, supporting the notion that lipid transfer may be a facet of PITP function in vivo.

scholarly journals BECLIN1: Protein Structure, Function and Regulation

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1522
Author(s):  
Sharon Tran ◽  
W. Douglas Fairlie ◽  
Erinna F. Lee
Keyword(s):  
Protein Structure ◽  
Structure Function ◽  
Membrane Trafficking ◽  
Biochemical Properties ◽  
Class Iii ◽  
Form Class ◽  
Protein Interactome ◽  
Protein Functions ◽  
Phosphoinositide 3 Kinase ◽  
Shed Light

BECLIN1 is a well-established regulator of autophagy, a process essential for mammalian survival. It functions in conjunction with other proteins to form Class III Phosphoinositide 3-Kinase (PI3K) complexes to generate phosphorylated phosphatidylinositol (PtdIns), lipids essential for not only autophagy but other membrane trafficking processes. Over the years, studies have elucidated the structural, biophysical, and biochemical properties of BECLIN1, which have shed light on how this protein functions to allosterically regulate these critical processes of autophagy and membrane trafficking. Here, we review these findings and how BECLIN1’s diverse protein interactome regulates it, as well as its impact on organismal physiology.

Two ubiquitin-like conjugation systems that mediate membrane formation during autophagy

2013 ◽  
Vol 55 ◽  
pp. 39-50 ◽  
Author(s):  
Hitoshi Nakatogawa
Keyword(s):  
Light Chain ◽  
Membrane Dynamics ◽  
The Other ◽  
Aminobutyric Acid ◽  
Amino Group ◽  
Autophagosome Formation ◽  
Acid Receptor ◽  
Receptor Associated Protein ◽  
Atg Proteins ◽  
C Terminus

In autophagy, the autophagosome, a transient organelle specialized for the sequestration and lysosomal or vacuolar transport of cellular constituents, is formed via unique membrane dynamics. This process requires concerted actions of a distinctive set of proteins named Atg (autophagy-related). Atg proteins include two ubiquitin-like proteins, Atg12 and Atg8 [LC3 (light-chain 3) and GABARAP (γ-aminobutyric acid receptor-associated protein) in mammals]. Sequential reactions by the E1 enzyme Atg7 and the E2 enzyme Atg10 conjugate Atg12 to the lysine residue in Atg5, and the resulting Atg12–Atg5 conjugate forms a complex with Atg16. On the other hand, Atg8 is first processed at the C-terminus by Atg4, which is related to ubiquitin-processing/deconjugating enzymes. Atg8 is then activated by Atg7 (shared with Atg12) and, via the E2 enzyme Atg3, finally conjugated to the amino group of the lipid PE (phosphatidylethanolamine). The Atg12–Atg5–Atg16 complex acts as an E3 enzyme for the conjugation reaction of Atg8; it enhances the E2 activity of Atg3 and specifies the site of Atg8–PE production to be autophagy-related membranes. Atg8–PE is suggested to be involved in autophagosome formation at multiple steps, including membrane expansion and closure. Moreover, Atg4 cleaves Atg8–PE to liberate Atg8 from membranes for reuse, and this reaction can also regulate autophagosome formation. Thus these two ubiquitin-like systems are intimately involved in driving the biogenesis of the autophagosomal membrane.

scholarly journals Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

2000 ◽  
Vol 347 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Brian S. FINLIN ◽  
Haipeng SHAO ◽  
Keiko KADONO-OKUDA ◽  
Nan GUO ◽  
Douglas A. ANDRES
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Cell Physiology ◽  
Gtp Binding ◽  
C Terminus ◽  
Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

scholarly journals Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

2020 ◽  
Author(s):  
Bishal Basak ◽  
Harini Krishnan ◽  
Padinjat Raghu
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Transfer Protein ◽  
Intramolecular Interaction ◽  
Lipid Transfer ◽  
Loss Of Function ◽  
Transfer Protein ◽  
Contact Sites ◽  
Accurate Localization ◽  
Phosphatidylinositol Transfer ◽  
Interdomain Interactions

Abstract During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.

scholarly journals Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates

2021 ◽  
Vol 8 ◽  
Author(s):  
Sabine Panzer ◽  
Chong Zhang ◽  
Tilen Konte ◽  
Celine Bräuer ◽  
Anne Diemar ◽  
...  
Keyword(s):  
Proton Pump ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Aureobasidium Pullulans ◽  
Green Light ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Proton Pumps ◽  
C Terminus ◽  
Pump Activity

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

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