Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIalpha at the trans-Golgi network.

Type II phosphatidylinositol 4-kinase IIα (PI4KIIα) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIα has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIα mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIα activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIα to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIα demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIα was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIα. Further measurements revealed that the reduction in the mobile fraction of PI4KIIα correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion.

associated proteins ( 45 ). However, it is not known how factors, such as membrane domain heterogeneity, TGN membrane continuity, or possible changes to PI4KII ␣ lateral diffusion, might contribute to the cholesterolsensitivity of the enzyme. Therefore, in this study we utilize a range of biochemical approaches together with spot photobleaching of enhanced green fl uorescent protein (eGFP)-tagged PI4KII ␣ to elucidate the mechanism underlying cholesterol-dependent PI4P synthesis.

Cell culture
Cells were maintained at 37°C in a humidifi ed incubator at 10% CO 2 . Cells were cultured in DMEM supplemented with Glutamax, 10% fetal calf serum, 50 i.u./ml penicillin and 50 g/ml streptomycin. For fl uorescence recovery after photobleaching (FRAP) measurements, cells were transfected 24 h prior to the experiments with eGFP-PI4KII ␣ .

Sonication-based assay to fragment PI4KII ␣ ؊ containing membranes
M ␤ CD (20 mM) was added to an equal volume of TGN membranes (usually 1 ml) on ice for 20 min to give a fi nal M ␤ CD concentration of 10 mM. Then 200 l of sodium carbonate 1M (pH 11.0) was added. The carbonate-treated membranes were probe sonicated followed by adjustment to 40% sucrose w/v in Tris-HCl 10 mM, EDTA 1 mM, and EGTA 1 mM (pH 7.4) to a fi nal volume of 4 ml. A discontinuous sucrose gradient was formed by overlaying the 40% sucrose layer with 4 ml of sucrose 30% w/v and 4 ml sucrose 5% w/v in Tris-HCl 10 mM, EDTA 1 mM, and EGTA 1 mM (pH 7.4). The gradient was centrifuged overnight at 185,000 g at 4°C and 1 ml fractions were harvested beginning at the top of the tube.

Immunoblotting of sucrose density gradient fractions
Equal volume aliquots of density gradient fractions were separated by SDS-PAGE, transferred to PVDF, and probed with anti-PI4KII ␣ or anti-syntaxin-6 antibodies. Western blots were quantifi ed using image analysis software in Adobe Photoshop CS4. about the membrane dynamics of PI4KII ␣ . Membrane lipid composition and, in particular, membrane cholesterol concentration underlie biophysical parameters such as membrane fl uidity, viscosity, and geometry, all of which are known to modulate membrane protein mobility ( 6 ). Cholesterol also has a role in the formation and organization of cholesterol-rich microdomains, often referred to as lipid rafts ( 11,12 ), which are proposed to organize membrane signaling events through transient and localized confi nement of the relevant signaling molecules ( 13 ). The trans -Golgi network (TGN) is particularly enriched in cholesterol (14)(15)(16) and also contains high levels of PI4P (17)(18)(19)(20). These observations have led some authors to suggest that PI4P synthesis is a determinant of TGN organelle identity ( 21,22 ). At the TGN, PI4KII ␣ activity is required for clathrin-dependent vesicle formation ( 3,23 ). Our main interest is in investigating the relationship between cholesterol and PI4P formation, particularly as the levels of both lipids are important for the formation of traffi cking vesicles at the TGN ( 3,(24)(25)(26) and our previous work demonstrated that PI4KII ␣ activity on intracellular membranes is dependent on membrane sterol levels ( 5 ).
The work presented here concerns TGN-associated PI4KII ␣ as opposed to the endosomal pool of the enzyme (27)(28)(29)(30)(31), which has been implicated in AP-3 adaptor recruitment ( 28,30,32 ). The TGN pool of PI4KII ␣ corresponds to the juxtanuclear and vesicular PI4KII ␣ pool that colocalizes with the TGN proteins syntaxin-6 ( 33 ) and TGN46 ( 34 ) both by microscopy ( 20,31,35 ) and by subcellular fractionation ( 36 ). PI4KII ␣ is unique among all the PI kinases in that it is constitutively associated with TGN membrane domains ( 20,(35)(36)(37) via posttranslational palmitoylation of a central CCPCC motif ( 35,38,39 ). PI4KII ␣ palmitoylation is essential for both kinase activity and for localization to the TGN, but this modifi cation is not absolutely required for membrane association of the enzyme ( 35,38,39 ). Here we focus on regulation of PI4KII ␣ at the TGN, with particular emphasis on the relationship between membrane organization and lipid composition in regulating PI4KII ␣ dynamics and activity.
Previously, we established that PI4KII ␣ and its phospholipid PI substrate associate with buoyant, TGN membrane domains ( 36 ) and that all the PI 4-kinase activity associated with these membranes was accounted for by the PI4KII ␣ isoform ( 5,31,36 ). Furthermore, we found that PI4P synthesis in these membrane preparations was sensitive to the manipulation of membrane sterol levels by methyl-␤ -cyclodextrin (M ␤ CD) ( 5 ). Cholesterol sequestration by M ␤ CD is often exploited experimentally to disrupt lipid rafts (reviewed in Ref. 40 ). Using this approach, other groups have shown that agonist-sensitive PI4P pools are M ␤ CD-sensitive ( 41 ) and that M ␤ CD delocalizes raft-associated PI4P ( 42 ). In addition to lipid raft disruption, cholesterol removal with M ␤ CD is known to induce a more condensed Golgi morphology ( 43 ), partial vesicularisation of TGN membranes ( 24,44 ), and changes in the lateral mobility of some TGN-selectively photobleached with 60 iterative rounds of illumination with the 488 nm line from a 30 mW Argon laser (Lasos Lasertechnik, Jena, Germany) set to 50% output and 100% transmission. Recovery of the fl uorescence signal in this spot was monitored by acquiring 12-bit images at 0.5 s intervals over a time course of 120 s, with the laser power reduced to 0. 9% transmission and with the pinhole fully open. To control unintentional photobleaching during the time course, an area from an adjacent cell with similar diameter and fl uorescence intensity was scanned in parallel without the initial photobleaching. Photobleaching during the time course was negligible, and it was not necessary to correct the FRAP data. All data were collected using a C-Apochromat 63×/1.2 water immersion objective (Carl Zeiss). For analysis of membrane connectivity, fl uorescence changes were monitored in an unbleached spot of equal area situated 3 M away from the photobleached spot used for FRAP analysis.
FRAP data were exported from Zeiss LSM 5 software and analyzed using GraphPad Prism 4 curve fi tting software. The diffusion coeffi cient (D) for eGFP-PI4KII ␣ was calculated as: e -2 .r 2 /4 1/2 where r is the radius of the photobleached spot and 1/2 is the half-time for maximal recovery of fl uorescence after photobleaching ( 48 ).
The mobile fraction of eGFP-PI4KII ␣ was calculated as: is the fl uorescence prior to photobleaching, F(0) refers to residual fl uorescence measured immediately after photobleaching, and F( ∞ ) is maximal fl uorescence recovery after photobleaching ( 48 ). Both F( ∞ ) and 1/2 were obtained by plotting fl uorescence against time in seconds and by nonlinear regression curve fi tting to a rectangular hyperbola desc ∞ ribed as: F = F( ∞ )·t/ 1/2 + t.

Cholesterol modulates PI substrate availability to PI4KII ␣
The PI 4-kinase activity associated with the buoyant TGN membrane fraction used in this study has been previously characterized and shown to be solely accounted for by PI4KII ␣ ( 5,31,36,37 ). In the absence of detergent, PI4KII ␣ in this membrane fraction can phosphorylate PI added in the form of small unilamellar vesicles (SUV) ( 48 ). We observed that M ␤ CD profoundly inhibited membrane-associated PI4P synthesis across a broad range (0-100 M) of added PI substrate ( Fig. 1A ). In the presence of M ␤ CD, PI4P synthesis was half-maximal at 3.5 M PI, which was not signifi cantly different from the halfmaximal concentration of PI measured in the absence of M ␤ CD ( 5,36 ). Therefore, M ␤ CD inhibited PI4KII ␣ without any apparent change in PI substrate affi nity. This raised the possibility that sterol depletion inhibited PI4P synthesis through effects on either protein diffusion and/ or membrane organization.

Non-cholesterol sterols and PI4KII ␣ activity
We previously demonstrated that the add-back of M ␤ CDcomplexed cholesterol can restore PI4P synthesis subsequent to cholesterol depletion by M ␤ CD ( 5 ). For the current study, we used M ␤ CD to replenish sterol-depleted PI4KII ␣rich membranes with noncholesterol sterols ( Fig. 1B ). The purpose of these experiments was to probe the molecular properties of sterols required to support intracellular

Determination of cholesterol levels
The cholesterol content of equal volume membrane fractions was assayed using the Amplex red cholesterol assay kit (Molecular Probes).

PI 4-kinase assays
PI 4-kinase assays using either endogenous membraneassociated PI or exogenous PI, and add-back of M ␤ CD complexed sterols were performed as previously described ( 5,31,36 ). Reaction products were separated by thin layer chromatography and visualized on a Typhoon 9400 phosphorimager (Amersham Biosciences). Quantitative data were obtained within the linear range of the instrument using ImageQuant Software (Amersham Biosciences). Specifi c PI4KII ␣ activity associated with each fraction was calculated by dividing the rate of PI4P generation (phosphorimager units/min) by the amount of PI4KII ␣ protein present in each fraction (arbitrary units) as determined by quantitation of anti-PI4KII ␣ Western blots. Data analysis and nonlinear regression curve fi tting were performed using Prism 5 software (GraphPad, San Diego, CA, USA) and compared using the Student t -test where significance was set at P < 0.05.

Fluorescence microscopy
Cells were grown on poly(L)lysine-coated glass coverslips for 24 h and then fi xed in 4% formaldehyde for 10 min on ice. Cells were permeabilized in 0.05% Triton X-100 for 5 min on ice, followed by immunostaining with anti-PI4KII ␣ . Cells were also directly stained, with or without the permeabilization step, using 50 g/ml fi lipin III (Sigma) for 20 min at room temp erature.
Filipin III-labeled samples were imaged using a Zeiss LSM 510 Meta laser-scanning confocal microscope system essentially as described ( 29 ) using 405 nm line for excitation of fi lipin III. We also used a wide-fi eld fl uorescence system consisting of a Leica DMIRB inverted microscope equipped with a heated chamber and an Imago QE CCD camera. Filipin III was excited using a Polychrome IV xenon arc light source tuned to 360 nm (Till Photonics GmbH, Gräfelfi ng, Germany). The use of this system reduced photobleaching of fi lipin III fl uorescence to negligible levels.
Imaging FRAP in eGFP-PI4KII ␣ -rich membranes COS-7 cells were grown on 35 mm glass-bottomed dishes (Wilco-dish, Intracel, Royston, Herts, UK) and transiently transfected 24 h prior to imaging, with a construct encoding eGFP-PI4KII ␣ ( 29 ). FRAP was performed on a Zeiss LSM 510 Meta equipped with a heated stage maintained at 37°C. Cells were sterol depleted by incubation with M ␤ CD for 20 min at 37°C in serum-free medium. Culture medium was replaced with Dulbecco's PBS containing 1 mM NaN 3 to completely inhibit the previously described intracellular traffi cking of eGFP-PI4KII ␣ -positive vesicles ( 29 ). The use of NaN 3 in FRAP experiments to distinguish between vesicular and nonvesicular traffi cking is well established ( 47 ). Furthermore, we found that while NaN 3 addition inhibited the movement of eGFP-PI4KII ␣ -containing vesicles, it did not induce any visible changes to the intracellular localization of eGFP-PI4KII ␣ . Therefore, NaN 3 addition permitted imaging of eGFP-PI4KII ␣ lateral diffusion without any fl uorescence changes caused by the intracellular traffi cking of PI4KII ␣ .
Prior to imaging FRAP, eGFP-PI4KII ␣ -rich membranes located in the juxtanuclear region were picked from randomly chosen transfectants (cells expressing high levels of eGFP-PI4KII ␣ were excluded). A circular area (0.7-1.0 m in diameter) was then zation, we evaluated the effects of M ␤ CD on PI4KII ␣ compartmentation within TGN membranes ( Fig. 2 ). As an assay for membrane microdomain heterogeneity, we used probe sonication to physically fragment a TGN-rich membrane fraction, followed by discontinuous equilibrium ultracentrifugation to separate the resultant membrane fragments on the basis of buoyant density ( 54,55 ). Our previous analyses revealed that sonication consistently results in the formation of vesicles of diameter 70-200 nm ( 37,56 ) and that this treatment also physically destroys any residual membrane network connec tivity. We investigated the effects of M ␤ CD on the distribution of cholesterol in the TGN gradient fractions. In gradients prepared from control membranes, cholesterol measured using the Amplex red cholesterol assay exhibited a broad asymmetric gradient distribution, with a main peak in gradient fraction 6 and gradually tailing off into the denser fractions ( Fig. 2A ). As expected, M ␤ CD reduced the overall sterol content of the membranes by 70%. However, most of this loss was from the buoyant broad peak of cholesterol in the 5-30% sucrose interface of the density gradient. The residual cholesterol was all localized to a minor pool peaking in fraction 9-11 of the gradient ( Fig. 2A ). We also investigated the effects of cholesterol depletion on the gradient distribution syntaxin-6, a protein that, similar to PI4KII ␣ , has been shown to associate with TGN microdomains ( 57, 58 ) ( Fig. 2B ). We found that the M ␤ CD addition resulted in a shift of syntaxin-6 protein from the 30% sucrose layer to the 30-40% sucrose interface. These results clearly show that M ␤ CD treatment resulted in a reduction in the sterol content and buoyancy of a subset of TGN membranes and that this density shift is most likely due to the increased protein:lipid ratio of the sterol-depleted fractions.
To investigate the relationship between PI4KII ␣ targeting to cholesterol-rich membranes and enzyme activity, we assayed PI4P synthesis using exogenous micellar PI substrate in the presence of TX-100 detergent. These assay conditions completely solubilize PI4KII ␣ ( 31 ), and endogenous membrane-associated PI is diluted to such an extent that it does not contribute toward the measured PI4P production ( 31 ). Therefore, these assay conditions report PI4KII ␣ activity outside the membrane environment. For control membranes (n = 3), the gradient distribution of PI4KII ␣ activity closely followed the distribution of PI4KII ␣ protein, with a broad peak between fractions 6 and 8 corresponding to the 30% sucrose layer ( Fig. 2C ). Treatment with M ␤ CD resulted in a marked loss of PI4KII ␣ protein from the 30% sucrose layer. In control gradients, 45.5 ± 3.6% (n = 3) of the total PI4KII ␣ protein was associated with the buoyant fraction, and subsequent to M ␤ CD addition, this was signifi cantly decreased to 11.7 ± 3.5% (n = 3). Loss of PI4KII ␣ protein was coupled with a loss of PI4KII ␣ activity from the buoyant fraction. However, sterol depletion did not result in an overall signifi cant decrease in the total amount of PI4KII ␣ activity measured across all the gradient fractions. Therefore, changes to microdomain PI4KII ␣ activity at the TGN. First we used M ␤ CD as a vehicle to substitute native membrane-associated cholesterol for its mirror-image molecule enantiomeric cholesterol, which does not occur naturally. Enantiomeric cholesterol ( 46,49,50 ) possesses the same physicochemical properties as cholesterol in model membranes but cannot effectively substitute for native cholesterol in processes involving direct protein:cholesterol interactions ( 46,51,52 ). We also investigated if membrane-associated PI4KII ␣ activity could be supported by desmosterol rather than cholesterol. Desmosterol is an immediate metabolic precursor of cholesterol that differs only by one double bond at the C24 position. Despite its structural similarity to cholesterol, desmosterol is much less effective in stabilizing liquid-ordered membrane domains ( 53 ). We found that both desmosterol and enantiomeric cholesterol could enhance [ 32 P]PI4P synthesis following sterol depletion ( Fig. 1B ). Furthermore, these data show that an enantiomeric or structurally specifi c cholesterol binding is not required for the activation of PI4KII ␣ , which instead depends on membrane biophysical properties.

Relationship between membrane microdomains and PI4KII ␣ localization
To explore the idea that inhibition of PI4KII ␣ activity by M ␤ CD was associated with a change in membrane organi- intracellular distribution of endogenous PI4KII ␣ by fl uorescence microscopy ( Fig. 3 ). Cholesterol was visualized on several different cellular membranes, including pronounced staining at the plasma membrane. PI4KII ␣ staining was more restricted to intracellular vesicles with very little staining at the plasma membrane. Our membrane fractionation data indicated that PI4KII ␣ distributed between membranes of differing cholesterol content. Differential colocalization of PI4KII ␣ with cholesterol was also observed in these imaging experiments, where both overlapping and nonoverlapping pools of PI4KII ␣ and cholesterol were visible in the juxtanuclear region of individual cells.
lipid composition delocalize PI4KII ␣ to a different biophysical environment but do not change the intrinsic activity of the enzyme. These results also demonstrate that removal of PI4KII ␣ from the membrane environment by detergent solubilization abrogates its sterol sensitivity, suggesting that PI4KII ␣ is only sterol-sensitive within the context of the native membrane.

Colocalization of PI4KII ␣ and cholesterol
Results from the density gradient assays demonstrated overlap between cholesterol and PI4KII ␣ -rich regions of the TGN. To validate this result, we imaged the distribution of cholesterol with fi lipin III and compared it to the Fig. 2. M ␤ CD alters the steady-state compartmentation of PI4KII ␣ . A TGN-enriched membrane fraction was probe sonicated and subfractionated in a discontinuous sucrose density gradient. A: The cholesterol content of equal volume aliquots from each gradient fraction was determined using the Amplex Red cholesterol assay. B: Anti-syntaxin 6 Western blot demonstrating the gradient distribution of syntaxin-6 in control and sterol-depleted membrane fractions. C: Gradient distribution of PI4KII ␣ protein and activity. Equal volume samples from each density gradient fraction were Western blotted and probed with an anti-PI4KII ␣ antibody. PI4KII ␣ activity associated with each fraction was determined by solubilizing the membranes and measuring [ 32 P]PI4P synthesis using a fi xed concentration of micellar PI. PI4KII ␣ protein levels were determined by quantitative analysis of anti-PI4KII ␣ Western blots. Specifi c PI4KII ␣ activity associated with each fraction was calculated by dividing the rate of PI4P generation (phosphorimager units.min Ϫ 1 ) by the amount of PI4KII ␣ protein present in each fraction as determined by Western blotting (arbitrary units). The results presented here are representative of three independent experiments. M ␤ CD, methyl-␤ -cyclodextrin; PI4KII, type II phosphatidylinositol 4-kinase; PI4P, phosphatidylinositol 4-phosphate.
replete cells, there was extensive overlap between eGFP-PI4KII ␣ and syntaxin-6, and this overlap was maintained following sterol depletion. In control cells, there was limited overlap between TGN46 and eGFP-PI4KII ␣ , and this partial colocalization was completely lost in the presence of M ␤ CD. These results demonstrate that the juxtanuclear pool of eGFP-PI4KII ␣ corresponds to a syntaxin-6-positive subdomain of the TGN. However, as syntaxin-6 has been localized to both the TGN and postTGN vesicles, it is more accurate to refer these membranes as a TGN-endosomal compartment.

FRAP experiments on cells expressing GFP-PI4KII ␣
As sterol depletion induced the redistribution of PI4KII ␣ to membrane compartments with differing biophysical properties and protein mobility can be modulated by membrane sterol content ( 6-10 ), we decided to investigate whether addition of M ␤ CD infl uenced PI4KII ␣ lateral diffusion. To measure the dynamics of PI4KII ␣ within cell membranes, FRAP experiments were carried out on COS-7 cells transiently transfected with GFP-PI4KII ␣ . We have previously determined that PI4KII ␣ -containing vesicles undergo rapid intracellular traffi cking ( 29 ). To differentiate between FRAP caused by membrane traffi cking events from that resulting solely from eGFP-PI4KII ␣ lateral diffusion, we carried out experiments in cells which had been azide-treated to deplete cellular ATP and thereby inhibit energy-dependent vesicular traffi cking ( 47 ). In these experiments, a spot of diameter 0.7-1.0 M was photobleached and fl uorescence recovery was measured at 0.5 s intervals over a 100-200 s time course. To standardize between different

M ␤ CD treatment affects the morphology of PI4KII ␣ -rich membranes
We investigated the effects of sterol depletion with M ␤ CD on the intracellular localization of PI4KII ␣ . In these experiments, we used confocal imaging to examine the effect of sterol depletion on eGFP-PI4KII ␣ and fi lipin III staining to image cholesterol. Under both control and cholesterol-depleted conditions, eGFP-PI4KII ␣ localized primarily to juxtanuclear intracellular vesicles ( Fig. 4 ). Addition of M ␤ CD induced two visible changes to the juxtanuclear pool of eGFP-PI4KII ␣ . First, the TGN pool of the enzyme contracted in size by up to 50%, and second, there was a visible vesicularization of the eGFP-PI4KII ␣ -containing membranes. These observations are consistent with the previously described effects of M ␤ CD on TGN membranes ( 24,43,44 ) . In addition, M ␤ CD treatment had dramatic effects on cellular sterol levels as imaged by fi lipin staining ( Fig. 4 ). Under these experimental conditions and notwithstanding some residual fi lipin staining in the juxtanuclear region, M ␤ CD treatment resulted in large losses of fi lipin staining throughout the cell.

Imaging the effects of M ␤ CD treatment on colocalization of PI4KII ␣ with TGN marker proteins
Our imaging of the intracellular distribution of eGFP-PI4KII ␣ following sterol depletion was consistent with M ␤ CD-induced changes to TGN morphology. This led us to consider the degree to which reductions in membrane sterol levels and subsequent changes to Golgi architecture might affect the colocalization of PI4KII ␣ with the TGN proteins TGN46 and syntaxin-6 ( Fig. 5 ). In cholesterol- Fig. 3. Distribution of sterol and endogenous PI4KII ␣ in A431 cells. Fixed cells were costained with fi lipin III (green channel) and an anti-PI4KII ␣ monoclonal antibody (red channel) and imaged by confocal microscopy. Arrows indicate membranes in the jutanuclear region with the greatest colocalization of fi lipin and PI4KII ␣ . Scale bars 10 m. Images are representative of three independent experiments. PI4KII, type II phosphatidylinositol 4-kinase.
Our analysis of the distribution of the juxtanuclear pool of eGFP-PI4KII ␣ demonstrated that the TGN-endosome membranes appeared more vesicular after addition of M ␤ CD. This led us to examine whether such a loss of membrane continuity could affect the dynamics of eGFP-PI4KII ␣ .We again used the spot photobleaching approach to determine the extent to which PI4KII ␣ was free to diffuse throughout the TGN-endosome compartment. To do this, we monitored fl uorescence changes in an identically sized but unbleached spot 3-4 m away from the photobleached spot ( Fig. 6C ). The reasoning behind this approach was that fl uorescence changes in the unbleached spot require exchange of eGFP-PI4KII ␣ with the photobleached area by lateral diffusion. Therefore, changes in fl uorescence in the unbleached spot report connectivity changes between different regions of the membrane. In control TGN membranes, we observed that fl uorescence loss and recovery in the unbleached spot closely followed the pattern of FRAP in the photobleached spot, although the magnitudes of such changes were reduced. However, addition of M ␤ CD resulted in a loss of fl uorophore movement between both areas, thereby revealing a loss of diffusion-based connectivity between both regions of the membrane. These results demonstrate that the reduction in overall recovery of eGFP-PI4KII ␣ subsequent to sterol depletion results from a marked loss of continuity in TGNendosomal membranes.

DISCUSSION
The key fi ndings of this study are that sterol depletion with M ␤ CD induces physical changes to TGNendosomal membranes, resulting in decreased PI4KII ␣ photobleaching experiments, we took care to choose spots of average fl uorescence intensity and always from eGFP-PI4KII ␣ -positive vesicular structures in the juxtanuclear region of the cells ( Fig. 6A ). Typically, photobleaching of control eGFP-PI4KII ␣ membranes resulted in a steady recovery of eGFP-PI4KII ␣ fl uorescence over a 2 min time course with a diffusion coeffi cient (D) of 0.87 ± 0.3 × 10 Ϫ 9 cm 2 .s Ϫ 1 (n = 6) and with 55.5 ± 11.2% (n = 6) of the protein mobile ( Fig. 6B ). We are confi dent that these measurements refl ect lateral diffusion since trial experiments revealed that the diffusion coeffi cient was linear with respect to the bleached spot area. However, for M ␤ CD-treated cells, the mobile fraction dropped to 30.9 ± 6.3% (n = 6) while D was increased at 2.4 ± 1.0 × 10 Ϫ 9 cm 2 .s Ϫ 1 (n = 6). The relatively large error in the calculated D value probably refl ects the heterogeneous effects of M ␤ CD on TGN morphology that we observed for the juxtanuclear eGFP-PI4KII ␣ compartment. It is has been reported that factors such as membrane topology and geometry can have a large effects on calculated D values ( 59,60 ). However, despite this complication, in four out of six paired experiments the apparent D value for eGFP-PI4KII ␣ was increased following M ␤ CD treatment. As regards the effects of M ␤ CD on the mobile fraction of eGFP-PI4KII ␣ , overall fl uorescence recovery was reduced by sterol depletion in six out of six paired experiments. Similarly, the half-time for fl uorescence recovery ( 1/2 ) on control membranes was 26.5 ± 10.8 s (n = 6) compared with 8.4 ± 3.5 s (n = 6) for steroldepleted membranes. In summary, our FRAP analysis revealed that M ␤ CD treatment induced signifi cant changes to the mobility of eGFP-PI4KII ␣ on TGN-endosomal membranes. cess ( 46,52,62 ). Therefore, the ability to restore PI4KII ␣ activity with enantiomeric cholesterol implies an indirect role for sterols in modulating PI4P synthesis through effects on membrane organization. We also found that even in instances where PI substrate is present in high concentrations, PI4KII ␣ activity was inhibited in the presence of M ␤ CD. This further strengthens the idea that cholesterol modulates PI4P synthesis indirectly by facilitating the interaction of PI4KII ␣ with its phospholipid substrate. Our membrane subfractionation studies demonstrated that M ␤ CD treatment led to a major loss of cholesterol from a pool of TGN-endosomal membranes, a concomitant collapse in membrane microdomain heterogeneity, and delocalization of PI4KII ␣ to denser membrane fractions. This shift in buoyant density is the fi rst evidence that a pool of PI4KII ␣ physically associates with cholesterol-rich membranes. Subsequent solubilization of the membrane fractions and assays for PI4P synthesis revealed that PI4KII ␣ intrinsic activity was not altered in cholesterol-depleted membranes. These data show that M ␤ CD-induced changes to membrane domain heterogeneity activity. Reduction in membrane cholesterol also induces a loss of TGN membrane continuity and a concomitant reduction in the PI4KII ␣ mobile fraction. This leads us to conclude that cholesterol-dependent compartmentation of PI4KII ␣ affects the dynamics of the enzyme and that this is important for PI4P generation. Note that our analyses of PI4KII ␣ distribution on steroldepleted membranes, both by membrane fractionation and by fl uorescence imaging, demonstrated that cholesterol is not absolutely required for association of the enzyme with the membrane. Furthermore, membrane association alone does not facilitate PI4P synthesis, as this is dependent on the membrane cholesterol content. In this regard, the ability of enantiomeric cholesterol to functionally substitute for native cholesterol in enhancing PI4P formation is a key fi nding. This is because cholesterol chirality does not affect spontaneous membrane domain formation or noncovalent interactions with other lipids ( 46,61 ). Cholesterol chirality is, however, important for cholesterolprotein binding, as this tends to be an enantiospecifi c pro- example, some have reported mobility differences between raft proteins and nonraft-associated proteins and suggested that proteins targeted to a single type of raft diffuse together as small entities ( 63,64 ). This differs from others who concluded that differences in protein mobility do not necessarily refl ect confi nement to lipid rafts ( 65 ). In the case of PI4KII ␣ , the differences in protein mobility that we observed can be rationalized by overall M ␤ CDinduced changes to membrane morphology and connectivity ( 24,43,44 ) without the need to invoke the targeting of PI4KII ␣ to lipid rafts. In particular, our photobleaching analysis demonstrated that M ␤ CD-induced changes to membrane structure limit the pool of fl uorophore available to replenish the photobleached spot, and this manifests as a reduced mobile fraction of eGFP-PI4KII ␣ . correlate with a decrease in productive interactions between PI4KII ␣ and PI. This is consistent with an important role for membrane cholesterol in maintaining PI4P generation through effects on membrane organization.
M ␤ CD treatment results in a decreased mobile fraction of eGFP-PI4KII ␣ , and this implies an important role for the TGN-endosomal cholesterol concentration in determining the overall mobility of the protein. While previous work demonstrated that a fraction of total cellular PI4KII ␣ associates with detergent-insoluble membrane domains ( 31 ), it does not necessarily mean that targeting of the protein-to-lipid rafts is the underlying explanation for the results we present here. Moreover, other studies have shown that the relationship between protein mobility and association with lipid rafts is not straightforward. As an