Abstract
BackgroundBiosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking.ResultsTo investigate the source of PI4P that orchestrates trafficking events, we developed a novel flow cytometry based method to monitor biosynthetic trafficking of transiently transfected proteins. We demonstrated that our method can be used to assess the trafficking of both type-1 transmembrane and GPI-linked proteins, and that it can accurately monitor the pharmacological disruption of biosynthetic trafficking with brefeldin A, a well-documented inhibitor of early biosynthetic trafficking. Furthermore, utilizing our newly developed method, we applied pharmacological inhibition of different isoforms of PI 4-kinase to reveal a role for a distinct pool of PI4P, synthesized by PI4KIIIα, in ER-to-PM trafficking.ConclusionsTaken together, these findings provide evidence that a specific pool of PI4P plays a role in biosynthetic trafficking of two different classes of proteins from the ER to the Golgi complex. Furthermore, our simple, flow cytometry-based biosynthetic trafficking assay can be widely applied to the study of multiple classes of proteins and varied pharmacological and genetic perturbations.
Highlights
Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles
To synchronize the early stages of biosynthetic trafficking, we developed a protocol in which Rat basophilic leukemia (RBL)-2H3 mast cells are incubated at 22°C for 14 hr following transient transfection with epidermal growth factor receptor (EGFR)-GFP
Representative confocal images of the suspended flow cytometry samples show that 6 hr after transfer to 37°C, EGFR is clearly detectable at the PM (Figure 1A)
Summary
Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking. Studies of the phosphorylated derivatives of phosphatidylinositol (PI) have shown that these molecules possess distinct biological functions and localize selectively to organelles (reviewed by [1]). Due to variable phosphorylation of hydroxyl groups on their inositol rings, seven different inter-convertible phosphoinositide species exist in cells, including PI4P and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 is a well-established regulator of multiple cellular processes, including vesicle trafficking [2], phagocytosis [3], membrane ruffling [4], cell motility and adhesion [5], and regulation of ion channel activity and receptor phosphorylation [6,7].
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