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

Abstract

PtdIns5P was discovered in 1997 [Rameh, Tolias, Duckworth and Cantley (1997) Nature 390, 192-196], but still 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. In the present paper we describe 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 1-2% of PtdIns4P levels. In the beta-pancreatic cell line BTC6, which is specialized in insulin secretion, PtdIns5P levels were higher than in most cells (2.5-4% 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 SER (smooth endoplasmic reticulum) 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 PIP4K (PtdIns5P 4-kinase) leads to accumulation of PtdIns5P in light fractions and fractions enriched in SER/Golgi, whereas 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)P(2) synthesis may play a role in Golgi-mediated vesicle trafficking.

FIGURE 1

PtdIns5P measurements using a novel HPLC method. (A) HPLC profile of [³H]inositol-labeled HeLa cells expressing IpgD. (B) HPLC profile of [³H]inositol-labeled HeLa cells transfected with control vector. (C) PtdIns5P levels (relative to total PI levels) in CHO-HIR cells before and after insulin stimulation (10 nM for 5 min), measured using [³²P]-labeled cellular lipids and the novel HPLC method for PtdIns5P detection. Data is the average and error bars are the standard error of three independent labelings. (D) HPLC profile of [³H]inositol-labeled HeLa treated with H₂O₂ (500 μM for 15 min). Insert shows PtdIns5P levels (relative to total PtdIns4P levels) in HeLa cells before and after H₂O₂ treatment. Data is the average and error bars are the standard error of four independent labelings.

FIGURE 2

Subcellular distribution of protein markers and lipids after differential centrifugation. Distribution of the membrane marker integrin β1 (white bars), the cytosolic proteins Erk 1/2 (hatched bars), the nuclear markers lamin A/C (HeLa cells) or phospho-histone (BTC6 cells) (grey bars) and total phosphoinositides (black bars) after differential centrifugation of BTC6 (A) or HeLa (B) cell lysates. S1 equals the sum of all 6 fractions from the sucrose gradient. The data shown represent the percentage distribution against the sum of all fractions and is representative of more than 3 separate experiments.

FIGURE 3

Distribution of protein markers and lipids after fractionation of the cytosolic/microsomal fraction through a 20%–65% sucrose gradient (A) Distribution of [³H]-total phosphoinositides from HeLa cells (solid line) detected after chloroform/methanol extraction of lipids from various fractions. Data shown is the average and range from two separate experiments. Also plotted is the sucrose density (dashed lines) measured from each fraction using a refractometer. (B and D) Distribution of the cytosolic protein Erk 1/2 (crosses) the Golgi protein βCOP (circles), the membrane protein integrin β1 (triangles) and the ribosomal protein S6 (squares) from HeLa cells (B) or BTC6 (D). (C) Distribution of the Golgi protein 58K (crosses), the membrane protein caveolin (circles), the mitochondrial protein ATP synthase (triangles) and the endosomal protein Rab5 (squares) from HeLa cells.

FIGURE 4

Distribution and relative concentration of PtdIns5P through the various subcellular fractions. [³H]-PtdIns5P (squares, solid line) and [³H]-PtdIns (stars), from HeLa (A and C) or BTC6 (B and D) cells were detected and quantified after chloroform/methanol extraction, deacylation and HPLC analysis of lipids from each fraction. In A and B, the amount of lipid present in each fraction is expressed as a percentage of its total from all fractions. In C and D, the concentration of PtdIns5P is expressed as percentage of PtdIns. Data shown is the average and standard error from 3 separate experiments. Due to the low lipid content in the sucrose fractions 1 and 6 and in the nuclear fraction of BTC6 cells (see Figures 2A and 4B), relative PtdIns5P determinations were subject to a larger error in these fractions.

FIGURE 5

Relative concentrations of PtdIns5P in each subcellular fraction in control or PIP4k IIγ knockdown HeLa cells. (A) Western blot showing the protein levels of PIP4k II β and PIP4k IIγ in control HeLa cells or HeLa cells infected with pSuper-IIα, pSuper-IIβ or pSuper-IIγ. Tubulin was used as loading control. (B) PtdIns5P levels in various subcellular fractions (relative to total phsphoinositide levels in each fraction) from HeLa control (black bars) or pSuper-IIγ infected HeLa cells (white bars). Data shown are the average and standard error of 3 (for control cells) and 4 (for PIP4k IIγ knockdown cells) independent experiments.

FIGURE 6

Distribution of phosphoinositides and relative concentration of PtdIns3P through the various subcellular fractions. [³H]-PtdIns3P (circles), [³H]-PtdIns4P (triangles), [³H]-PtdIns(4,5)P₂ (diamonds) and [³H]-PtdIns (stars) were detected after chloroform/methanol extraction of lipids from each fraction, deacylation and HPLC analysis. In A and B the amount of lipid present in each fraction is expressed as a percentage of its total from all fractions. In C and D, the concentration of PtdIns3P is expressed as percentage of PtdIns. Data shown is the average and standard error from 3 separate experiments.

FIGURE 7

Distribution of protein markers for ER, Golgi and endosomes after fractionation of the BTC6’s cytosolic/microsomal fraction through a 20%–65% sucrose gradient (A) and effect of Brefeldin A on PtdIns5P and PtdIns3P distribution (B). (A) Distribution of the early endosomal protein EEA1 (stars) and Rab5 (open circles), the COPI protein βCOP (black circles), the Golgi marker GM130 (black squares), the ER protein calnexin (triangles) and PIP4k IIγ (diamonds) from BTC6 cell lysates. The amount of each protein present in each fraction is expressed as a percentage of its total from all fractions. (B) Distribution of PtdIns5P (squares) and PtdIns3P (circles) before (black squares and circles) or after (open square and circles) treatment with Brefeldin A (2 μg/ml for 2 hrs). The amount of each lipid present in each fraction is expressed as a percentage of its total from all fractions. Data is the average and bars represent the standard error of two experiments (except for GM130 which is from one experiment).