A novel HPLC-based approach makes possible the spatial characterization of cellular PtdIns5P and other phosphoinositides

Abstract

SYNOPSIS Phosphatidylinositol-5-phosphate (PtdIns5P) was discovered in 1997 (Rameh, L.E.; Tolias, K.F.; Duckworth, B.C. and Cantley, L.C. 1997, Nature 390, 192–196) but very little is known about its regulation and function. Hitherto, studies of PtdIns5P regulation have been hindered by the inability to measure cellular PtdIns5P using conventional HPLC, owing to poor separation from PtdIns4P. Here we present a new HPLC method for resolving PtdIns5P from PtdIns4P, which makes possible accurate measurements of basal and inducible levels of cellular PtdIns5P in the context of other phosphoinositides. Using this new method, we found that PtdIns5P is constitutively present in all cells examined (epithelial cells, fibroblasts and myoblasts, among others) at levels typically 1% to 2 % of PtdIns4P levels. In the β-pancreatic cell line BTC6, which is specialized in insulin secretion, PtdIns5P levels were higher than in most cells (2.5% to 4% of PtdIns4P). Using subcellular fractionation, we found that the majority of the basal PtdIns5P is present in the plasma membrane, but it is also enriched in intracellular membrane compartments, especially in smooth endoplasmic reticulum (SER) and/or Golgi, where high levels of PtdIns3P were also detected. Unlike PtdIns3P, PtdIns5P was also found in fractions containing very low-density vesicles. Knockdown of PtdIns5P 4-kinase (PIP4k) leads to accumulation of PtdIns5P in light fractions and fractions enriched in SER/Golgi, while treatment with Brefeldin A results in a subtle, but reproducible, change in PtdIns5P distribution. These results indicate that basal PtdIns5P and the PtdIns5P pathway for PtdIns(4,5)P2 synthesis may play a role in Golgi-mediated vesicle trafficking.

Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates TOR signaling and cell growth during Drosophila development

“…3 B and C). Consistent with the idea that adult body size in insects is predetermined during larval development we observed that newly eclosed adult dPIP4K 29 flies weighed less than the wild-type controls (Fig. 3H).…”

“…This revealed that PI5P levels in dPIP4K 29 were twice that measured in wild-type controls (Fig. 2D); by contrast, the levels of PI4P (Fig.…”

“…3A). The maximum difference in body weight was at 60 h posthatching (at 22°C) when dPIP4K 29 larvae weighed approximately 54% of controls; toward the end of larval development this difference is relatively reduced (dPIP4K 29 larvae weigh approximately 85% of wild-type controls) but with a delay of approximately 12 h (Fig. 3A).…”

“…2F). We reconstituted dPIP4K 29 with either a wildtype dPIP4K transgene or the point mutant allele [amino acid 271 (D271K) was mutated from aspartic acid to lysine, Fig. 1C] that is kinase dead.…”

Structural Basis for Interactions of thePhytophthora sojaeRxLR Effector Avh5 with Phosphatidylinositol 3-Phosphate and for Host Cell Entry

“…Just like other phosphoinositides, PtdIns(5)P also has its own subcellular locations. The majority of PtdIns(5)P is localized in the plasma membrane, while the minor part is in Golgi and Sarco/endoplasmic reticulum (SER) markers (57,58). It has been suggested that PtdIns(5)P plays crucial roles in cellular signaling pathways, although the corresponding functional role needs to be well defined (46).…”