Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non- palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.Author Summary: The endoplasmic reticulum (ER) is the largest intracellular organelle of mammalian cells. It is responsible for many fundamental cellular functions, such as folding, quality control of membrane and secreted protein, lipid biosynthesis, control of apoptosis and calcium storage. Recent studies have shown that many ER membrane proteins are lipid modified. We therefore hypothesized that palmitoyltransferases, the enzymes responsible for this modifications, act as a regulator of the mammalian ER, controlling the function of a network of key proteins through reversible acylation. In this work we combine computational methods with experimental determination of parameters to study the mechanisms and properties of ER palmitoylation, using as a model the palmitoylation of the ER protein calnexin. The systematic analysis of the mathematical model, built and calibrated with the help of experimental data, shows that Calnexin palmitoylation leads to a 9-fold increase in half-life and that a long delay separates synthesis from palmitoylation in unstimulated cells. Surprisingly during this delay, 75% of synthesized calnexin is degraded before being palmitoylated. We hypothesize that this unexpected apparent inefficiency is a design principle that provides the cell with a means to post-translationally tune the calnexin content.