A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIα is important for protein trafficking from the endoplasmic reticulum to the plasma membrane

Bryant et al. BMC Cell Biology A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase III is important for protein trafficking from the endoplasmic reticulum to the plasma membrane Kirsten L Bryant 0 1 Barbara Baird 1 David Holowka 1 0 University of North Carolina , Chapel Hill, NC 27514 , USA 1 Department of Chemistry and Chemical Biology, Cornell University , Ithaca, NY , USA Background: 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. Results: To 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 PI4KIII, in ER-to-PM trafficking. Conclusions: Taken 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. Biosynthetic protein trafficking; Phosphoinositide 4-phosphate; Flow cytometry - Background 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). Different phosphoinositide species are often enriched in distinct intracellular membranes; for example, PI(4,5)P2 is predominately localized to the inner leaflet of the PM, whereas PI4P is enriched at the Golgi complex. 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]. In addition, PI(4,5)P2 is the substrate for generation of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [8], and thereby is necessary for agonist-stimulated Ca2+ signaling. Furthermore, there have been several reports of PI(4,5)P2 existing in functionally and spatially distinct pools in the PM that support specific signaling platforms [9-12]. PI4P, the most prevalent mono-phosphorylated PI-derivative in cells [13], was for many years believed to serve no function outside of being the precursor for PIP2 [14]. Recently, however, a number of PI4P-dependent processes have been characterized, in particular its role in the regulation of protein trafficking. For example, PI4P strongly promotes COPII-mediated export of proteins at endoplasmic reticulum (ER) exit sites (ERES; [15,16]). Also, by interacting with the lipid transfer proteins CERT, OSBP, and FAPP (collectively termed COFs), PI4P plays roles in sphingolipid and sterol biosynthetic trafficking [17]. Organelle-specific phosphoinositide distributions are maintained by the tight regulation of PI-kinases and PI-phosphatases. Four distinct PI 4-kinases have been described in mammalian cells, including type II (PI4KII and PI4KII) and type III (PI4KIII and PI4KIII) kinases [18]. The type II PI 4-kinases are palmitoylated [19] and thus strongly membrane associated, particularly in the trans-Golgi apparatus [20], and, to a lesser extent, in endosomes [21]. PI4KIII localizes primarily to the Golgi apparatus, coincident with Arf1, a small GTP-binding protein [22,23]. Although the molecular details of how these enzymes are linked to Golgi-derived biosynthetic transport remain unknown, they have all been implicated in Golgi function and secretion [21]. Deletion of the gene for PI4KIII is embryonically lethal in mice [24], and its normal subcellular distribution is complex, with evidence for cytosolic [24], PM [24,25], and ER [22] concentration. Recently, PI4KIII has been identified as a critical host factor for hepatitis C viral replication [26]. With regard to biosynthetic trafficking, PI4P localized to the Golgi apparatus has been implicated in the delivery of cargo from the Golgi to the PM [27,28], in addition to evidence supporting a role for PI4P in COPII nucleation at ERES cited above [15,16]. The present study addresses PI4P participation in ER-to-Golgi trafficking. Using a novel technique to monitor protein biosynthetic trafficking, we show that pharmacological inhibition of PI4KIII results in ER-retention of both the epidermal growth factor receptor (EGFR) and a glycophosphatidylinositol (GPI)-anchored protein. Furthermore, we show that inhibition of a Golgi-localized PI 4-kinase does not result in ER-retention. Taken together, these findings provide evidence that a specific pool of PI4P, synthesized by PI4KIII, is essential for biosynthetic protein trafficking. Results Flow cytometry of transiently transfected cells to assess perturbations of early ER-to-Golgi trafficking events Preliminary experiments indicated that testing the effects of potential inhibitors of ER-to-Golgi trafficking requires a method in which a substantial percentage of transfected protein makes this transition simultaneously. To synchronize the early stages of biosynthetic trafficking, we developed a protocol in which RBL-2H3 mast cells are incubated at 22C for 14 hr following transient transfection with EGFR-GFP. This incubation at lower temperature allows for protein synthesis to occur with minimal trafficking to the PM [29,30]. The next day, the cells are transferred to 37C, at which time biosynthetic trafficking begins. Cells are harvested and fixed at the various time points (depending on the experiment), and unpermeabilized cells are labeled with an antibody specific for an extracellular epitope. Thus, the increased presence of EGFR-GFP at the PM can be quantified over time by flow cytometry by measuring the total EGFR-GFP fluorescence and comparing it to the fluo (...truncated)