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.
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Research Article|
September 14 2011
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)
Kathryn Garner;
Kathryn Garner
1Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6JJ, U.K.
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Michelle Li;
Michelle Li
1Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6JJ, U.K.
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Natalie Ugwuanya;
Natalie Ugwuanya
1Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6JJ, U.K.
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Shamshad Cockcroft
Shamshad Cockcroft
1
1Department of Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6JJ, U.K.
1To whom correspondence should be addressed (email S.cockcroft@ucl.ac.uk).
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Publisher: Portland Press Ltd
Received:
April 11 2011
Revision Received:
June 21 2011
Accepted:
June 28 2011
Accepted Manuscript online:
June 28 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 439 (1): 97–111.
Article history
Received:
April 11 2011
Revision Received:
June 21 2011
Accepted:
June 28 2011
Accepted Manuscript online:
June 28 2011
Citation
Kathryn Garner, Michelle Li, Natalie Ugwuanya, Shamshad Cockcroft; 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). Biochem J 1 October 2011; 439 (1): 97–111. doi: https://doi.org/10.1042/BJ20110649
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