Abstract
BackgroundIn mammalian cells protein-lipid interactions at the trans-Golgi network (TGN) determine the formation of vesicles, which transfer secretory proteins to the cellular membrane. This process is regulated by a complex molecular network including protein kinase D (PKD), which is directly involved in the fission of transport vesicles, and its interaction with the ceramide transfer protein CERT that transports ceramide from the endoplasmic reticulum to the TGN.ResultsHere we present a novel quantitative kinetic model for the interactions of the key players PKD, phosphatidylinositol 4-kinase III beta (PI4KIII β) and CERT at the TGN membranes. We use sampling-based Bayesian analysis and perturbation experiments for model calibration and validation.ConclusionsOur quantitative predictions of absolute molecular concentrations and reaction fluxes have major biological implications: Model comparison provides evidence that PKD and CERT interact in a cooperative manner to regulate ceramide transfer. Furthermore, we identify active PKD to be the dominant regulator of the network, especially of CERT-mediated ceramide transfer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0147-1) contains supplementary material, which is available to authorized users.
Highlights
In mammalian cells protein-lipid interactions at the trans-Golgi network (TGN) determine the formation of vesicles, which transfer secretory proteins to the cellular membrane
Data-driven modeling of protein kinase D (PKD), PI4KIIIβ and Ceramide transfer protein (CERT) at the TGN The exact mechanism by which CERT transfers ceramide from the endoplasmic reticulum (ER) to the TGN is currently still unclear [6,10,13]
The ’neck-swinging’ model suggests that CERT is simultaneously bound to both membranes, while extracting ceramide from the ER membranes via its Steroidogenic acute regulatory protein (START) domain and releasing it at the TGN membrane [2,39]
Summary
In mammalian cells protein-lipid interactions at the trans-Golgi network (TGN) determine the formation of vesicles, which transfer secretory proteins to the cellular membrane. Cargo is received from the ER at the cis-Golgi complex, traverses the medial Golgi stacks, reaches the transGolgi side where vesicles bud off from the trans-Golgi network (TGN) [2,3] The formation of these vesicles is driven by a complex interaction of proteins and lipids and is still only partly understood. The first TGN-localized PKD substrate identified was PI4KIIIβ whose phosphorylation on serine 294 increased the activity of the lipid kinase, resulting in the enhanced production of its substrate PI4P [7] This phosphorylated lipid is especially important in defining Golgi membrane identity and providing a local signaling platform to which several PI4P-binding proteins are recruited, including the ceramide transfer protein CERT [8,9]
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