Biology subjects

Doyle, C. P.

Publications and source records attributed to Doyle, C. P..

4 recordsLinked to original sources

bioRxiv2023

OSBP is a major determinant of Golgi phosphatidylinositol 4-phosphate homeostasis

The lipid phosphatidylinositol 4-phosphate (PI4P) plays a master regulatory role at Golgi membranes, orchestrating membrane budding, non-vesicular lipid transport and membrane organization. It follows that harmonious Golgi function requires strictly maintained PI4P homeostasis. One of the most abundant PI4P effector proteins is the oxysterol binding protein (OSBP), a lipid transfer protein that exchanges trans Golgi PI4P for ER cholesterol. Although this protein consumes PI4P as part of its lipid anti-porter function, whether it actively contributes to Golgi PI4P homeostasis has been questioned. Here, we employed a series of acute and chronic genetic manipulations, together with orthogonal targeting of OSBP, to interrogate its control over Golgi PI4P abundance. Modulating OSBP levels at ER:Golgi membrane contact sites produces reciprocal changes in PI4P levels. Additionally, we observe that OSBP has a high capacity for PI4P turnover, even at orthogonal organelle membranes. However, despite also visiting the plasma membrane, endogenous OSBP makes no impact on PI4P levels in this compartment. We conclude that OSBP is a major determinant of Golgi PI4P homeostasis.

cell biology↗

bioRxiv2023

Orthogonal targeting of SAC1 to mitochondria implicates ORP2 as a major player in PM PI4P turnover

Oxysterol binding protein (OSBP)-related proteins (ORPs) 5 and 8 have been shown to deplete the lipid phosphatidylinositol 4-phosphate (PI4P) at sites of membrane contact between the endoplasmic reticulum (ER) and plasma membrane (PM). This is believed to be caused by transport of PI4P from the PM to the ER, where PI4P is degraded by an ER-localized SAC1 phosphatase. This is proposed to power the anti-port of phosphatidylserine (PS) lipids from ER to PM, up their concentration gradient. Alternatively, ORPs have been proposed to sequester PI4P, dependent on the concentration of their alternative lipid ligand. Here, we aimed to distinguish these possibilities in living cells by orthogonal targeting of PI4P transfer and degradation to PM-mitochondria contact sites. Surprisingly, we found that orthogonal targeting of SAC1 to mitochondria enhanced PM PI4P turnover independent of targeting to contact sites with the PM. This turnover could be slowed by knock-down of soluble ORP2, which also has a major impact on PM PI4P levels even without SAC1 over-expression. The data reveal a role for contact site-independent modulation of PM PI4P levels and lipid antiport.

cell biology↗

bioRxiv2022

Depletion of Plasma Membrane PI4P by ORP5 Requires Hydrolysis by SAC1 in Acceptor Membranes

The authors have withdrawn their manuscript because the central conclusion is incorrect. We discovered this while revising the paper after peer review. In the original submission, we described ectopic targeting of the PI4P transfer protein, ORP5. We reported that when combined with orthogonal targeting of SAC1 to the mitochondrial outer membrane, ORP5 facilitates depletion of PM PI4P when targeted to PM-mitochondria contact sites. New experiments revealed that orthogonal targeting of SAC1 to the mitochondria alone is sufficient to deplete PM PI4P; ORP5 is not required. Thus, although the data reported in the manuscript are valid and reproducible, the conclusion was incorrect. We are currently performing additional experiments to better characterize and understand the effects of mitochondrially-targeted SAC1 on PM pools of PI4P. We will post a new manuscript detailing these findings on bioRXiv, which will be submitted for peer review at the original journal as a revision. In the meantime, we are withdrawing the preprint so as not to mislead the field with the erroneous conclusion. We sincerely apologize to anyone whose work was misdirected by our honest mistake. Therefore, the authors do not wish this work to be cited as reference for the project in its present form. If you have any questions, please contact the corresponding author.

cell biology↗

bioRxiv2022

A Novel Homeostatic Mechanism Tunes PI(4,5)P2-dependent Signaling at the Plasma Membrane

The lipid molecule phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes that synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions may drive disease associated with disrupted PI(4,5)P2 homeostasis. One-Sentence SummaryThe enzyme PIP4K functions as both a sensor and negative regulator of PI(4,5)P2 synthesis by the closely related PIP5K enzymes, tuning the activity of numerous membrane functions.

cell biology↗