- Email: [email protected]
Subcellular localisation of inositol lipid kinases in rat liver
Biochimica et Biophysica Acta 845 (1985) 163-170
163
Elsevier BBA 11459
Subcellular Iocalisation of inositol lipid kinases in rat liver
Shamshad Cockcroft, Janet A. Taylor and Jacob D. Judah Department of Experimental Pathology, School of Medicine, University College, University St., London, WC1E 6JJ (U.K.)
(ReceivedAugust28th, 1984) (Revised manuscriptreceivedJanuary28th, 1985)
Key words: Inositollipid kinase; Subcellularlocalization;(Rat liver)
The subceilular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is Iocalised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.
Introduction The polyphosphoinositides constitute a minor fraction of the phospholipids in most cells, but it is now clear that they are of considerable metabolic significance [1,2]. Their phosphate groups are capable of rapid turnover, because of the presence of specific phosphokinases and of phosphatases. Our interest is in the phosphokinases, one of which, in the presence of ATP, transfers phosphate groups to phosphatidylinositol (PtdIns) to yield phosphatidylinositol 4-phosphate (PtdIns4P). This molecule is capable of a second phosphorylation (by another phosphokinase) to yield phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2), which may be of key importance in the regulation of cell function. Its breakdown, brought about by phospholipase C in response to CaE+-mobilising hormones, yields diacylglycerol and inositol trisphosphate, both implicated in second messenger functions [2]. Diacylglycerol activates protein kinase C and there is evidence that inositol trisphosphate is concerned with the mobilisation of Ca 2+ from internal stores. Because of this association between receptor activation and inositol lipid breakdown it has been
assumed that the polyphosphoinositides (and by implication, the kinases that phosphorylate PtdIns) are localised at the plasma membrane [1-3] but this localisation is estalished only for the mammalian erythrocyte, which in any event contains no internal membranes [4]. Contrary to the above assumption, previous studies on the distribution of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase have produced conflicting results. The plasma membrane [5-8], the Golgi [9,10], secretory granules [8,11,12], endoplasmic reticulum [9], lysosomes and mitochondria [14] have all been suggested as possible sites for phosphatidylinositol kinase, and plasma membrane [7], cytosol [15] and Golgi [9] for phosphatidylinositol-4-phosphate kinase. To resolve this inconsistency we have studied the subcellular localisation of the inositol lipid kinases in rat liver using the endogenous lipid as substrate. We show that the enzyme that phosphorylates PtdIns to PtdIns4P is present in Golgi membranes and lysosomes as well as in the plasma membrane, whilst the second kinase that phosphorylates PtdIns4P to PtdIns(4,5)P2 is found only in the plasma membrane.
0167-4889/85/$03.30 © 1985 ElsevierScience Publishers B.V. (BiomedicalDivision)
164 by the method of Butler and Judah [23]. Marker enzymes were assayed as in Taylor et al. [19].
M a t e r i a l s and M e t h o d s
Phosphatidylinositol was bought from the Sigma Chemical Co Ltd, Poole, Dorset, U.K. y-labelled [ 32P]ATP was synthesised by the enzymatic method of Glynn and Chappell [16] and its specific activity was measured by high-pressure liquid chromatography as described previously [17].
Fractionation of rat liver In order to prepare the various fractions with a high degree of enrichment, different fractionation procedures were used to optimise the purification of the individual components. Overall recoveries in a complete set of fractions was performed for the Golgi preparation (Table I). The homogenised liver was subjected to a low-speed spin to obtain a nuclear pellet (also containing most of the mitochondria and lysosomes). The supernatant was then spun to obtain a microsomal pellet and the cytosol. The microsomal fraction was then subjected to density-gradient centrifugation to obtain the light Golgi fractions (GF~+2) [18] and further purified on Ficoll gradients (when required) by the method of Taylor et al. [19]. Plasma membranes were prepared by the method of Dorling and Le Page [20]. Smooth and rough endoplasmic reticulum fractions were prepared on sucrose gradients [21]. Lysosomes loaded with Triton WR1339 were prepared as described by Leighton et al. [22]. Mitochondria were isolated
Determination of phosphokinase activity Assays were carried out in 100 #1 of reaction mixture which contained 1 mM [32 P]ATP (specific radioactivity 10-50 c p m / p m o l ) , 20 m M Mg 2÷, 50 m M Tris-HC1, 125 m M sucrose, 0.5 m M E G T A and approx. 50-200 #g membrane protein at p H 7. For experiments with plasma membrane fractions 2 mM ATP was used. Incubation temperature was 30°C, and the reaction was started by adding 50 /~1 M g . ATP 2 to 50 /~1 of membrane suspension. The reaction was stopped after 1 min (except where the time-course was being studied) by addition of 1.5 ml c h l o r o f o r m / m e t h a n o l / c o n c . HC1 ( 2 0 : 4 0 : 1 , by vol.) and the resulting singlephase system was partitioned by addition of 0.5 ml each of chloroform and distilled water. The lower (chloroform) phase from each sample was washed with the upper phase obtained after partitioning a mixture of c h l o r o f o r m / m e t h a n o l / 1 M HC1 ( 1 : 1 : 0 . 9 , by vol). The washed lower phase was evaporated to dryness and redissolved in chloroform (50 #1). In those experiments where ATP levels were measured the reaction was terminated by addition of 2 M HCIO 4 and the lipids extracted from the resulting precipitate as described above; the supernatant was retained for ATP analysis [171. The lipids were separated by one-dimensional
TABLE I LOCALISATION OF INOSITOL LIPID KINASES IN RAT LIVER GOLGI Enzyme assays were carried out on various subcellular fractions and starting homogenate as described in the Materials and Methods section. Results are expressed either as a mean (+ S.D.) from three or more experiments or data from individual experiments where only two experiments were done. Number in brackets relates to the number of experiments, n.d., not detected. Fraction
Spec. act. of Ptdlns kinase (pmol 32p in
% Recoveryof PtdIns kinase
Spec.act. % Recoveryof of PtdIns4P kinase Ptdlns4P kinase
activity
(pmol 32p in PtdIns(4,5) P2/min per mg protein)
activity
38.5 + 6 (3) 40 + 1 (3) 6.4+ 2 (3)
19, 25 31, 42 17, 25 11, 15
(100) 43, 57 11, 15 7, 16
5.8 +0.9 (8) 18, 42 1.7, 4.6 n.d.
(100) 65 36 n.d.
6.7+1 (3)
84, 237
0.4, 1.2
595+107 (8)
11.7+1.6%(3)
Ptdlns4P/min per mg protein) Homogenate
Microsomes Nuclear pellet Cytosol Golgi GF1+/
48+ 13 (3) 218 + 58 (3) 37+ 12 (3) 19 + 6 (3) 2609+160(3)
(100)
Spec. act. of galactosyltransferase (nmol [ 3H]galactose in
% Recovery of galactosyltransferase
ovalbumin/h per mg protein)
165
provide the substrate. Therefore the presence of the second kinase may be missed in those fractions where the first kinase is absent.
TLC on oxalate-impregnated plates using chlorog o r m / m e t h a n o l / a c e t o n e / acetic a c i d / w a t e r (40 : 13 : 15 : 12 : 8, by vol.) and the plate was subsequently subjected to autoradiography. Radioactivity in the individual lipids was determined as described [17]. In most experiments the endogenous Ptdlns of the membrane fraction acted as acceptor. In experiments where the Golgi was further purified on Ficoll gradients exogenous Ptdlns was added as acceptor at 1 mM in the presence of 0.25% (w/v) Triton X-100 as described by Collins and Wells [13]. All assays were performed in duplicate which did not vary by more than 10%.
Results
In preliminary experiments we tried varying concentrations of Mg 2÷ (1-20 mM) and ATP (0.5-2 mM) to establish optimal conditions for phosphatidylinositol kinase activity in the Golgi fraction. This was established to be 20 mM for Mg z+ and 1 mM for ATP for maximal activity. The conditions used here are similar to those described by Jergil and Sundler [10] and Collins and Wells [13]. The activities of the inositol lipid kinases were sought in smooth and rough endoplasmic reticulum, in a light Golgi fraction (GF1- 2), in a plasma membrane fraction, in lysosomes, in mitochondria and in the cytosol.
Calculation of the results
The number of pmol of lipid synthesised was calculated from the specific activity of the ATP. For the formation of Ptdlns4P it was assumed that 1 mol of phosphate was incorporated per mol of Ptdlns4P formed and for the formation of Ptdlns(4,5)P 2 it was assumed that 2 mol of phosphate were incorporated per mol of lipid. The amount of phosphorylated lipid formed may be underestimated because no attempt was made to control the activity of phosphatases which may be p r e s e n t . M o r e o v e r , the c a l c u l a t i o n for Ptdlns(4,5)P 2 formed is also based on the assumption that no endogenous Ptdlns4P was available. Another parameter that needs to be considered is the availability of substrate for the kinase. For phosphatidylinositol kinase substrate will not be rate-limiting, since Ptdlns is present in sufficient amounts. However, this will certainly not be the case for phosphatidylinositol-4-phosphate kinase. Instead it is dependent on the first kinase to
Golgi fraction G F1 + 2
Phosphatidylinositol kinase is concentrated in the Golgi (Table I). It is enriched 54-fold in relation to the parent homogenate, with a recovery of 7%. In the same preparation galactosyltransferase is enriched approx. 100-fold, with a 12% recovery. Since the transferase is uniquely localised in the Golgi [24,25] it may be inferred that about 50% of the phosphatidylinositol kinase is to be found in the Golgi fraction of a liver homogenate. The Golgi fraction displays little activity of phosphatidylinositol-4-phosphate kinase, which will be referred to later when we consider the results with plasma membrane fractions. The G F t + 2 was subjected to centrifugation on a
Goloctosyl " L t r°nsfer'°se 4F
•~-
6I 3
o
PI kinase
:I /
r"
Protein
2.0
oL#
1.5
u
1.C
L L ~
0.5 0 L-
1.;2 1.~)4 1.;6 1.081 Density
I
1
I I / I 1.02 1.04 1.06 1.08 Density
Density
Fig. 1. Distribution of enzymes after centrifugation of GFI+ 2 on Ficoll gradients. 1-ml samples were taken from the gradients, and enzyme and protein analyses carried out as described in the Materials and Methods section. Density was determined by refractometry and relative concentration calculated by dividing the activity in each sample by the activity which would be in that fraction if distribution were uniform throughout the whole gradient.
166 linear Ficoll gradient. T h e h i s t o g r a m in Fig. 1 shows that m o s t of the activity of p h o s p h a t i d y l i n o s i t o l kinase c o m i g r a t e s with that of galactosyltransferase. A smaller fraction s e d i m e n t s further d o w n the g r a d i e n t a n d coincides with a p e a k of h e x o s a m i n i d a s e activity a n d is p r o b a b l y due to l y s o s o m a l c o n t a m i n a t i o n of the G F I + 2 fraction (see later). Fig. 2 shows the time-course of the f o r m a t i o n of P t d l n s 4 P in the G o l g i m e m b r a n e s . T h e rate is linear for a p p r o x . 6 m i n a n d progress halts soon thereafter. This is n o t due to e x h a u s t i o n either of A T P or of P t d l n s . T h e b r e a k d o w n of A T P b y the G o l g i p r e p a r a t i o n , s t u d i e d in two experiments, was a p p r o x . 35 n m o l A T P / m i n p e r m g p r o t e i n at 30°C, so that even after 10 rain a b o u t 65% o f the A T P was still intact. T h e G o l g i m e m b r a n e c o n t a i n s 60 n m o l P t d l n s / m g p r o t e i n [19], b u t o n l y 5 n m o l of P t d l n s 4 P were f o r m e d at the p l a t e a u of the progress curve. It m a y be that o n l y 8% of the P t d l n s was available for p h o s p h o r y l a t i o n to a c c o u n t for this result, b u t it is i m p o s s i b l e f r o m these d a t a to rule out the activity o f a p h o s p h a t a s e in the m e m b r a n e .
The plasma membrane fraction F r o m T a b l e II it is clear that the kinase converting P t d l n s to P t d l n s 4 P is well r e p r e s e n t e d in the p l a s m a m e m b r a n e , with an e n r i c h m e n t of s o m e 30-fold a n d a recovery of 15%. This is very similar to the e n r i c h m e n t a n d recovery of 5 ' - n u c l e o t i d a s e ( T a b l e II). However, we are u n a b l e to use these d a t a to e s t i m a t e w h a t p r o p o r t i o n of the p h o s -
5
D
o
4--
c
~ 3 ~2 9 c
0
I
I
I
[
I
I
0
2
4
6
8
10
Min
Fig. 2. Time-course of Ptdlns4P and Ptdlns(4,5)P2 formation in rat liver Golgi. Golgi membranes were incubated in the presence of ATP (1 raM) and samples withdrawn into acidified chloroform:methanol at the appropriate times. The lipids were extracted and separated on thin-layer chromatography. The amount of lipid synthesised was calculated from the amount of label incorporated into the lipids and the specific activity of the ATP. Open circles, nmol Ptdlns4P formed per mg protein, and closed circles, nmol PtdIns(4,5)P 2 formed per mg protein.
p h a t i d y l i n o s i t o l k i n a s e activity is localised at the p l a s m a m e m b r a n e b e c a u s e in rat liver only a p prox. 50% of the 5 ' - n u c l e o t i d a s e is localised in this fraction [26]. T h e kinase r e s p o n s i b l e for the f o r m a t i o n of P t d l n s ( 4 , 5 ) P 2 is seen ( T a b l e II) to b e l o c a t e d a l m o s t exclusively in the p l a s m a m e m b r a n e fraction, where it is enriched 30-fold. The l y s o s o m e s
TABLE II DISTRIBUTION OF INOSITOL LIPID KINASES IN VARIOUS MEMBRANE FRACTIONS OF RAT LIVER Legend as in Table I. Abbreviations: RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum, n.d., not determined. Fraction
Spec. act. of Ptdlns kinase (pmo132p in Ptdlns4P/min per mg protein)
% Recovery of Ptdlns kinase activity
Spec. act. % Recovery of of Ptdlns4P Ptdlns4P kinase kinase (pmol 32p activity in Ptdlns(4,5) P2/min per mg protein)
Spec. act. of 5'-nucleotidase (#mol AMP hydrolysed/h per mg protein)
% Recovery 5'-nucleotidase
Homogenate RER SER
132, 94 71, 120 141, 228
(100) 1, 1 1.6, 1.2
11.5, 5.6 14, 14 28, 42
(100) 2.6, 2.1 3.8, 3.8
n.d. n.d. n.d.
n.d. n.d. n.d.
Homogenate Plasma membrane
79 + 14 (4) 2642 + 320 (6)
(100) 12, 18
32 + 11 (3) 954 + 250 (6)
(100) 9, 4
1.2 _+1.3 (6) 26.5 + 1.8 (5)
(100) 17 _+2.1
167
and the Golgi membranes contain little of the enzyme and that small activity seen in the Golgi could well be due to contamination by plasma membrane fragments. Fig. 3A shows the progress of labelling of Ptdlns4P and of Ptdlns(4,5)P 2 in plasma membranes in the presence of [32p]ATP. The rate of formation of Ptdlns4P is linear for only 2 min and declines slowly thereafter; the rate of formation of Ptdlns(4,5)P 2 is slow compared to that of Ptdlns4P. In all experiments there was an actual decline with time (over a 10 min period) in the amount of the two labelled species (data not shown). This was coincidental with the nearly total disappearance of ATP from the reaction mixture measured at 10 min (data not shown). Fig. 3B shows the rate of decline of ATP in the experiment shown in Fig. 3A. The activity of the ATPase in five separate experiments was 286 + 53 nmol A T P / m i n per mg protein at 30 °. It was therefore essential to keep the membrane addition to no more than 1 m g / m l in the reaction mixture, to add ATP to 2 mM instead of 1 mM and to use short incubation times. However, the breakdown of ATP produces another problem. Since the ATP is y-labelled, breakdown yields unlabelled ADP which in turn is converted to unlabelled ATP by
5
IO0
4
BO o~ c 60 c_
c
o. 3
the action of adenylate kinase (confirmed by HPLC). This means that the specific radioactivity of the ATP falls with time. In turn, the calculation of the formation of PtdIns4P, and more partaiculady that of PtdIns(4,5)P2, will be underestimated if the reaction is allowed to run for too long. Bearing this in mind, it is calculated that of the 40 nmol P t d I n s / m g protein in the plasma membrane fraction [19], some 7 nmol at the very least was converted to polyphosphoinositides. The method of Dorling and Le Page [20] used to prepare plasma membranes has present in the homogenising medium 0.5 mM C a 2+. Since this may activate a polyphosphoinositide phosphodiesterase [27], it may affect the phosphokinase assay in two ways: the generation of diacylglycerol may affect enzyme activity or the endogenous level of polyphosphoinositides would be depleted and thus alter the activity of inositol lipid kinases. To make sure that the presence of Ca 2÷ in the initial homogenising medium was not affecting the activity of either of the two inositol lipid kinases we prepared plasma membranes in the absence of C a 2+. This greatly reduces the yield but does not affect the qualitity of the preparation. We found that the activity of both the inositol lipid kinases (phosphatidylinositol kinase, 2628 p m o l / m i n per mg protein; phosphatidylinositol-4-phosphate kinase 1073 p m o l / m i n per mg protein) were similar to that shown in Table II for membranes prepared with Ca 2÷.
2
a. 2o ~ B E 0 c
0 0
3 Min
4
0
1
2
3 Min.
4
5
Fig. 3. A. Time-course of PtdIns4P and PtdIns(4,5)P 2 formation in rat liver plasma m e m b r a n e fraction. Assays were conducted as described in the legend of Fig. 2 except that the reactions were terminated by addition of 1 M perchloric acid. The precipitated protein was extracted for lipids as described in Fig. 2. The supernatants were retained for determination of ATP. Open circles, nmol of PtdIns4P formed per mg protein, and closed circles, nmol PtdIns(4,5)P 2 formed per mg protein in rat liver plasma membranes. B. Time-course of A T P hydrolysis by rat liver plasma m e m b r a n e fraction. Assays were performed on supernatants obtained after protein precipitation as described in A. supernatants were neutralised with K O H and the a m o u n t of A T P was determined by HPLC.
c_ o ci
(3. _L Q_
3-
2
1 E c
I
0
I
2
I
,4
I
6
I
~.
I 10
Min.
Fig. 4. Time-course of Ptdlns4P formation in rat hver lysosomes. Assay of phosphatidylinositol kinase was conducted as described in the legend to Fig. 2.
168
Endoplasmic reticulum
Mitochondria
As shown in Table I, microsomes of rat liver contain substantial amounts of both the phosphokinases under study, though at a low specific activity, and one may conclude that most of the activity is due to their content of Golgi and plasma membrane fragments. This is confirmed by the isolation of purified smooth and rough endoplasmic reticulum (Table II), which shows that activity is low compared to Golgi and plasma membrane.
The mitochondrial fraction showed negligible activity of the inositol lipid kinases, with specific activities well below those of the original homogenate (data not shown).
Lysosomes Collins and Wells [13] have reported that lysosomes of rat liver can phosphorylate phosphatidylinositol in the presence of ATP. Neither the enrichment nor the recovery of the enzyme activity in the lysosomal fraction was reported, however. Table III shows the results of our experiments with Triton-loaded lysosomes. The enrichment (29-fold) and the recovery (3%) of the kinase compares with 67-fold enrichment and 8% recovery of hexosaminidase and suggests that the kinase does indeed have a significant representation in lysosomes. Only the Golgi or plasma membrane fractions could contribute such large activities of the kinase, but marker enzymes for these (galactosyltransferase or 5'-nucleotidase and alkaline phosphodiesterase) show very tittle contamination by Golgi or plasma membrane fragments (data not shown). Negligible activity was detected for phosphatidylinositol-4-phosphate kinase (data not shown).
Cytosol Phosphatidylinositol-4-phosphate kinase activity has been identified as a cytosolic enzyme in brain [15]. To test the possibility that the cytosol may also contain this enzyme we prepared Golgi fraction (GF 1+2) and incubated this with cytosol in the presence of [32p]ATP. The presence of the cytosol had no effect on phosphatidylinositol kinase activity and on the level of Ptdlns4P formed and neither was any Ptdlns4P converted into Ptdlns(4,5)P 2, thus ruling out the presence of any cytosolic activity that was capable of converting the substrate presented by the membrane. Discussion In contrast to the endoplasmic reticulum, which is the principal location of phospholipid biosynthetic activity in rat liver [28], the synthesis of the polyphosphoinositides from Ptdlns occurs in a variety of cell-membranes. Downes and Michell [1] have reviewed the metabolism of the polyphosphoinositides and have discussed the data on the intracellular localisation of the kinases responsible for their synthesis from Ptdlns (see also Refs. 28 and 29 for reviews). Michell et al. [5] reported that phosphatidylinositol-4-phosphate kinase was con-
TABLE 1II LOCALISATION OF INOSITOL LIPID KINASES IN RAT LIVER LYSOSOMES Legend as in Table I. The specificactivityof phosphatidylinositol-4-phosphatekinase in the lysosomaifraction was negligible(5 pmol 32p in Ptdlns(4,5)P2/min per mg protein). Fractions
Homogenate Large granules Lysosomes
Spec. act. of Ptdlns kinase (pmol 32p in Ptdlns4P /rain per mg protein) 53+ 9 (4) 66+ 8(4) 1476+205(4)
% Recoveryof Ptdlns kinase activity
(100) 12.6+2.7 3 +1
Spec. act. of hexoseaminidase (# mol 2-nitrophenol formed/h per mg protein) 1.6+0.5 (6) 3.6+0.6(8) 105 +3.4(8)
% Recoveryof hexoseaminidase activity
(100) 30+7 8+2
169 centrated in the plasma membrane of rat liver. Phosphatidylinositol kinase has also been found in the Golgi apparatus [10] and in lysosomes [131. The present study confirms that the enzymes responsible for phosphorylation of Ptdlns are present in the plasma membranes, the Golgi apparatus and to some extent in the lysosomes of rat liver. We also report the distribution of the phosphatidylinositol-4-phosphate kinase. Because of the exclusive localisation of phosphatidylinositol-4phosphate kinase in the plasma membrane, it may be inferred that Ptdlns(4,5)P2 would be a reliable marker for the plasma membrane (providing that phospholipid exchange proteins do not transport it to other intracellular membranes). It may be inferred, too, that the breakdown of Ptdlns(4,5)P 2 in response to hormones must also be at the plasma membrane, generating diacylglycerol and inositol trisphosphate. Since the latter is released to the cytosol, it may be concluded that the lipid must be localised to the inner bilayer of the plasma membrane. It has been suggested that the phosphatidylinositol kinase is confined to Golgi membranes alone [10] but this is clearly not the case. The Golgi preparation is able to phosphorylate only 8% of its endogenous Ptdlns. This figure agrees with that found by Jergil and Sundler [10]. This degree of phosphorylation may reflect the amount of Ptdlns accessible to its enzyme. However, in the presence of Triton X-100, Jergil and Sundler [10] reported that this figure could be increased to only 20%. Thus it is very likely that product inhibition sets the limit as to how much Ptdlns can be phosphorylated in the Golgi, since in the plasma membrane, where Ptdlns4P is removed by further phosphorylation, more of the Ptdlns gets phosphorylated (see below). In the plasma membrane at least 17% of Ptdlns can be converted into polyphosphoinositides. The major contaminant of the plasma membrane preparation is the endoplasmic reticulum and it can be calculated from the specific activity of glucose6-phosphatase that it accounts for 20% of the preparation, as protein. Taking into account that endoplasmic reticulum has twice the amount of Ptdlns compared to plasma membrane [19], then the amount of Ptddlns that gets phosphorylated in the plasma membrane increases to 30%. As outlined in the Methods and Discussion the amount
of polyphosphoinositides formed in the plasma membrane will be underestimated so that the 30% is the absolute minimum. Thus the interesting possibility is raised that the major proportion of the inositol lipid in plasma membranes is present as the phosphorylated derivatives of Ptdlns. Such a situation does occur in the erythrocyte where the only membrane is a plasma membrane [31-33]. Phosphatidyhnositol kinase is known to be present in secretory granules of chromaffin cells [11], parotid [12] and the pancreatic fl-cell [8]. We presume that its presence in the Golgi apparatus of liver is analogous to these. However, the function of the enzyme in all four systems is obscure and may have some role in secretion, which is under hormonal control in chromaffin cells, the parotid and the pancreas, but not in the liver, which apparently produces and secretes the plasma proteins continuously. One possibility is that the phosphorylation of Ptdlns helps to provide a negative charge on the membrane which may be altered in the presence of Ca 2÷. Since Ca 2+ is known to inhibit the kinase [8], it is possible that the rise in cytosolic Ca 2÷ following cell activation observed in many cells may allow dephosphorylation to occur and the removal of the charge may allow membranes to come together before they fuse. The presence of Ptdlns kinase in lysosomes may play a similar role. The presence of both inositol lipid kinases in the plasma membrane is not surprising, since PtdIns(4,5)P 2 is implicated in the generation of two putative second messengers [2].
Acknowledgements We thank the Medical Research Council, The Wellcome Trust and the Vandervell Trust for grants in aid of our work.
References 1 Downes, C.P. and Micheli, R.H. (1982) Cell Calcium 3, 467-502 2 Berridge, M.J. (1984) Biochem.J. 220, 345-360 3 Michell, R.H. (1979) Trends Biochem. Sci. 4, 128-131 4 Hokin, L.E. and Hokin, M.R. (1964) Biochim. Biophys. Acta 84, 563-566 5 Michell,R.H., Harwood,T.L., Coleman, R. and Hawthorne, J.N. (1967) Biochim. Biophys.Acta 144, 649-658 6 Kai, M., White, G.L. and Hawthorne,J.N. (1966) Biochem. J. 101, 328-337
170 7 Rawyler, A.J., Roelofsen, B., Wirtz, K.W.A. and Op den Kamp, J.A.F. (1982) Febs Lett. 148, 140-144 8 Tooke, N.E., Hales, C.N. and Hutton, J.C. (1984) Biochem. J. 219, 471-480 9 Cooper, P.H. and Hawthorne, J.N. (1976) Biochem. J. 160, 97-105 10 Jergil, B. and Sundler, R. (1983) J. Biol. Chem. 258, 7968-7973 11 Phillips, J.H. (1973) Biochem. J. 136, 579-587 12 Oron, Y., Sharoni, Y., Lefkovitz, H. and Selinger, Z. (1978) in Cyclitols and Phosphoinositides (Wells, W.W. and Eisenberg, J.F., eds.), pp. 383-397, Academic Press, New York 13 Collins, C.A. and Wells, W.W. (1983) J. Biol. Chem. 258, 2130-2134 14 Hajra, A.K., Seguin, E.B. and Agranoff, B.W. (1968) J. Biol. Chem. 243, 1609-1616 15 Kai, M., Salway, J.G. and Hawthorne, J.N. (1976) Biochem. J. 106, 791-801 16 Glynn, I.M. and Chappell, J.B. (1964) Biochem. J. 90, 147-149 17 Cockcroft, S. (1984) Biochim. Biophys. Acta 795, 37-46 18 Bergeron, J.J.M. (1979) Biochim. Biophys. Acta 55, 493-503 19 Taylor, J.A., Limbrick, A.R., Allan, D. and Judah, J.D. (1984) Biochim. Biophys. Acta 769, 171-178 20 Dorling, P.R. and Le Page, R.N. (1973) Biochim. Biophys. Acta 318, 33-44
21 Gelter, D.M., Judah, J.D. and Nicholls, M.R. (1972) Biochem. J. 127, 865-874 22 Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S. and De Duve, C. (1968) J. Cell Biol. 37, 482-513 23 Butler, W.H. and Judah, J.D. (1969) J. Cell Biol. 44, 278-289 24 Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45-72 25 Farquhar, M.G., Bergeron, J.J.M. and Palade, G.E. (1974) J. Cell Biol. 60, 8-25 26 Stanley, K.K., Edwards, M.R. and Luzio, J.P. (1980) Biochem. J. 186, 59-69 27 Cockcroft, S., Baldwin, J.M. and Allan, D. (1984) Biochem. J. 221,477-482 28 Jelsema, C.L. and Morre, D.J. (1978) J. Biol. Chem. 253, 7960-7971 29 Michell, R.H. (1975) Biochim. Biophys. Acta 415, 81-147 30 Hawthorne, J.N. (1983) Biosci. Reps. 3, 887-904 31 Peterson, S.C. and Kirschner, L.B. (1970) Biochim. Biophys. Acta 202, 295-304 32 Schneider, R.P. and Kirschner, L.B. (1970) Biochim. Biophys. Acta 202, 283-294 33 Ferrell, J.E., Jr. and Huestis, W.H. (1984) J. Cell Biol. 98, 1992-1998
163
Elsevier BBA 11459
Subcellular Iocalisation of inositol lipid kinases in rat liver
Shamshad Cockcroft, Janet A. Taylor and Jacob D. Judah Department of Experimental Pathology, School of Medicine, University College, University St., London, WC1E 6JJ (U.K.)
(ReceivedAugust28th, 1984) (Revised manuscriptreceivedJanuary28th, 1985)
Key words: Inositollipid kinase; Subcellularlocalization;(Rat liver)
The subceilular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is Iocalised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.
Introduction The polyphosphoinositides constitute a minor fraction of the phospholipids in most cells, but it is now clear that they are of considerable metabolic significance [1,2]. Their phosphate groups are capable of rapid turnover, because of the presence of specific phosphokinases and of phosphatases. Our interest is in the phosphokinases, one of which, in the presence of ATP, transfers phosphate groups to phosphatidylinositol (PtdIns) to yield phosphatidylinositol 4-phosphate (PtdIns4P). This molecule is capable of a second phosphorylation (by another phosphokinase) to yield phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2), which may be of key importance in the regulation of cell function. Its breakdown, brought about by phospholipase C in response to CaE+-mobilising hormones, yields diacylglycerol and inositol trisphosphate, both implicated in second messenger functions [2]. Diacylglycerol activates protein kinase C and there is evidence that inositol trisphosphate is concerned with the mobilisation of Ca 2+ from internal stores. Because of this association between receptor activation and inositol lipid breakdown it has been
assumed that the polyphosphoinositides (and by implication, the kinases that phosphorylate PtdIns) are localised at the plasma membrane [1-3] but this localisation is estalished only for the mammalian erythrocyte, which in any event contains no internal membranes [4]. Contrary to the above assumption, previous studies on the distribution of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase have produced conflicting results. The plasma membrane [5-8], the Golgi [9,10], secretory granules [8,11,12], endoplasmic reticulum [9], lysosomes and mitochondria [14] have all been suggested as possible sites for phosphatidylinositol kinase, and plasma membrane [7], cytosol [15] and Golgi [9] for phosphatidylinositol-4-phosphate kinase. To resolve this inconsistency we have studied the subcellular localisation of the inositol lipid kinases in rat liver using the endogenous lipid as substrate. We show that the enzyme that phosphorylates PtdIns to PtdIns4P is present in Golgi membranes and lysosomes as well as in the plasma membrane, whilst the second kinase that phosphorylates PtdIns4P to PtdIns(4,5)P2 is found only in the plasma membrane.
0167-4889/85/$03.30 © 1985 ElsevierScience Publishers B.V. (BiomedicalDivision)
164 by the method of Butler and Judah [23]. Marker enzymes were assayed as in Taylor et al. [19].
M a t e r i a l s and M e t h o d s
Phosphatidylinositol was bought from the Sigma Chemical Co Ltd, Poole, Dorset, U.K. y-labelled [ 32P]ATP was synthesised by the enzymatic method of Glynn and Chappell [16] and its specific activity was measured by high-pressure liquid chromatography as described previously [17].
Fractionation of rat liver In order to prepare the various fractions with a high degree of enrichment, different fractionation procedures were used to optimise the purification of the individual components. Overall recoveries in a complete set of fractions was performed for the Golgi preparation (Table I). The homogenised liver was subjected to a low-speed spin to obtain a nuclear pellet (also containing most of the mitochondria and lysosomes). The supernatant was then spun to obtain a microsomal pellet and the cytosol. The microsomal fraction was then subjected to density-gradient centrifugation to obtain the light Golgi fractions (GF~+2) [18] and further purified on Ficoll gradients (when required) by the method of Taylor et al. [19]. Plasma membranes were prepared by the method of Dorling and Le Page [20]. Smooth and rough endoplasmic reticulum fractions were prepared on sucrose gradients [21]. Lysosomes loaded with Triton WR1339 were prepared as described by Leighton et al. [22]. Mitochondria were isolated
Determination of phosphokinase activity Assays were carried out in 100 #1 of reaction mixture which contained 1 mM [32 P]ATP (specific radioactivity 10-50 c p m / p m o l ) , 20 m M Mg 2÷, 50 m M Tris-HC1, 125 m M sucrose, 0.5 m M E G T A and approx. 50-200 #g membrane protein at p H 7. For experiments with plasma membrane fractions 2 mM ATP was used. Incubation temperature was 30°C, and the reaction was started by adding 50 /~1 M g . ATP 2 to 50 /~1 of membrane suspension. The reaction was stopped after 1 min (except where the time-course was being studied) by addition of 1.5 ml c h l o r o f o r m / m e t h a n o l / c o n c . HC1 ( 2 0 : 4 0 : 1 , by vol.) and the resulting singlephase system was partitioned by addition of 0.5 ml each of chloroform and distilled water. The lower (chloroform) phase from each sample was washed with the upper phase obtained after partitioning a mixture of c h l o r o f o r m / m e t h a n o l / 1 M HC1 ( 1 : 1 : 0 . 9 , by vol). The washed lower phase was evaporated to dryness and redissolved in chloroform (50 #1). In those experiments where ATP levels were measured the reaction was terminated by addition of 2 M HCIO 4 and the lipids extracted from the resulting precipitate as described above; the supernatant was retained for ATP analysis [171. The lipids were separated by one-dimensional
TABLE I LOCALISATION OF INOSITOL LIPID KINASES IN RAT LIVER GOLGI Enzyme assays were carried out on various subcellular fractions and starting homogenate as described in the Materials and Methods section. Results are expressed either as a mean (+ S.D.) from three or more experiments or data from individual experiments where only two experiments were done. Number in brackets relates to the number of experiments, n.d., not detected. Fraction
Spec. act. of Ptdlns kinase (pmol 32p in
% Recoveryof PtdIns kinase
Spec.act. % Recoveryof of PtdIns4P kinase Ptdlns4P kinase
activity
(pmol 32p in PtdIns(4,5) P2/min per mg protein)
activity
38.5 + 6 (3) 40 + 1 (3) 6.4+ 2 (3)
19, 25 31, 42 17, 25 11, 15
(100) 43, 57 11, 15 7, 16
5.8 +0.9 (8) 18, 42 1.7, 4.6 n.d.
(100) 65 36 n.d.
6.7+1 (3)
84, 237
0.4, 1.2
595+107 (8)
11.7+1.6%(3)
Ptdlns4P/min per mg protein) Homogenate
Microsomes Nuclear pellet Cytosol Golgi GF1+/
48+ 13 (3) 218 + 58 (3) 37+ 12 (3) 19 + 6 (3) 2609+160(3)
(100)
Spec. act. of galactosyltransferase (nmol [ 3H]galactose in
% Recovery of galactosyltransferase
ovalbumin/h per mg protein)
165
provide the substrate. Therefore the presence of the second kinase may be missed in those fractions where the first kinase is absent.
TLC on oxalate-impregnated plates using chlorog o r m / m e t h a n o l / a c e t o n e / acetic a c i d / w a t e r (40 : 13 : 15 : 12 : 8, by vol.) and the plate was subsequently subjected to autoradiography. Radioactivity in the individual lipids was determined as described [17]. In most experiments the endogenous Ptdlns of the membrane fraction acted as acceptor. In experiments where the Golgi was further purified on Ficoll gradients exogenous Ptdlns was added as acceptor at 1 mM in the presence of 0.25% (w/v) Triton X-100 as described by Collins and Wells [13]. All assays were performed in duplicate which did not vary by more than 10%.
Results
In preliminary experiments we tried varying concentrations of Mg 2÷ (1-20 mM) and ATP (0.5-2 mM) to establish optimal conditions for phosphatidylinositol kinase activity in the Golgi fraction. This was established to be 20 mM for Mg z+ and 1 mM for ATP for maximal activity. The conditions used here are similar to those described by Jergil and Sundler [10] and Collins and Wells [13]. The activities of the inositol lipid kinases were sought in smooth and rough endoplasmic reticulum, in a light Golgi fraction (GF1- 2), in a plasma membrane fraction, in lysosomes, in mitochondria and in the cytosol.
Calculation of the results
The number of pmol of lipid synthesised was calculated from the specific activity of the ATP. For the formation of Ptdlns4P it was assumed that 1 mol of phosphate was incorporated per mol of Ptdlns4P formed and for the formation of Ptdlns(4,5)P 2 it was assumed that 2 mol of phosphate were incorporated per mol of lipid. The amount of phosphorylated lipid formed may be underestimated because no attempt was made to control the activity of phosphatases which may be p r e s e n t . M o r e o v e r , the c a l c u l a t i o n for Ptdlns(4,5)P 2 formed is also based on the assumption that no endogenous Ptdlns4P was available. Another parameter that needs to be considered is the availability of substrate for the kinase. For phosphatidylinositol kinase substrate will not be rate-limiting, since Ptdlns is present in sufficient amounts. However, this will certainly not be the case for phosphatidylinositol-4-phosphate kinase. Instead it is dependent on the first kinase to
Golgi fraction G F1 + 2
Phosphatidylinositol kinase is concentrated in the Golgi (Table I). It is enriched 54-fold in relation to the parent homogenate, with a recovery of 7%. In the same preparation galactosyltransferase is enriched approx. 100-fold, with a 12% recovery. Since the transferase is uniquely localised in the Golgi [24,25] it may be inferred that about 50% of the phosphatidylinositol kinase is to be found in the Golgi fraction of a liver homogenate. The Golgi fraction displays little activity of phosphatidylinositol-4-phosphate kinase, which will be referred to later when we consider the results with plasma membrane fractions. The G F t + 2 was subjected to centrifugation on a
Goloctosyl " L t r°nsfer'°se 4F
•~-
6I 3
o
PI kinase
:I /
r"
Protein
2.0
oL#
1.5
u
1.C
L L ~
0.5 0 L-
1.;2 1.~)4 1.;6 1.081 Density
I
1
I I / I 1.02 1.04 1.06 1.08 Density
Density
Fig. 1. Distribution of enzymes after centrifugation of GFI+ 2 on Ficoll gradients. 1-ml samples were taken from the gradients, and enzyme and protein analyses carried out as described in the Materials and Methods section. Density was determined by refractometry and relative concentration calculated by dividing the activity in each sample by the activity which would be in that fraction if distribution were uniform throughout the whole gradient.
166 linear Ficoll gradient. T h e h i s t o g r a m in Fig. 1 shows that m o s t of the activity of p h o s p h a t i d y l i n o s i t o l kinase c o m i g r a t e s with that of galactosyltransferase. A smaller fraction s e d i m e n t s further d o w n the g r a d i e n t a n d coincides with a p e a k of h e x o s a m i n i d a s e activity a n d is p r o b a b l y due to l y s o s o m a l c o n t a m i n a t i o n of the G F I + 2 fraction (see later). Fig. 2 shows the time-course of the f o r m a t i o n of P t d l n s 4 P in the G o l g i m e m b r a n e s . T h e rate is linear for a p p r o x . 6 m i n a n d progress halts soon thereafter. This is n o t due to e x h a u s t i o n either of A T P or of P t d l n s . T h e b r e a k d o w n of A T P b y the G o l g i p r e p a r a t i o n , s t u d i e d in two experiments, was a p p r o x . 35 n m o l A T P / m i n p e r m g p r o t e i n at 30°C, so that even after 10 rain a b o u t 65% o f the A T P was still intact. T h e G o l g i m e m b r a n e c o n t a i n s 60 n m o l P t d l n s / m g p r o t e i n [19], b u t o n l y 5 n m o l of P t d l n s 4 P were f o r m e d at the p l a t e a u of the progress curve. It m a y be that o n l y 8% of the P t d l n s was available for p h o s p h o r y l a t i o n to a c c o u n t for this result, b u t it is i m p o s s i b l e f r o m these d a t a to rule out the activity o f a p h o s p h a t a s e in the m e m b r a n e .
The plasma membrane fraction F r o m T a b l e II it is clear that the kinase converting P t d l n s to P t d l n s 4 P is well r e p r e s e n t e d in the p l a s m a m e m b r a n e , with an e n r i c h m e n t of s o m e 30-fold a n d a recovery of 15%. This is very similar to the e n r i c h m e n t a n d recovery of 5 ' - n u c l e o t i d a s e ( T a b l e II). However, we are u n a b l e to use these d a t a to e s t i m a t e w h a t p r o p o r t i o n of the p h o s -
5
D
o
4--
c
~ 3 ~2 9 c
0
I
I
I
[
I
I
0
2
4
6
8
10
Min
Fig. 2. Time-course of Ptdlns4P and Ptdlns(4,5)P2 formation in rat liver Golgi. Golgi membranes were incubated in the presence of ATP (1 raM) and samples withdrawn into acidified chloroform:methanol at the appropriate times. The lipids were extracted and separated on thin-layer chromatography. The amount of lipid synthesised was calculated from the amount of label incorporated into the lipids and the specific activity of the ATP. Open circles, nmol Ptdlns4P formed per mg protein, and closed circles, nmol PtdIns(4,5)P 2 formed per mg protein.
p h a t i d y l i n o s i t o l k i n a s e activity is localised at the p l a s m a m e m b r a n e b e c a u s e in rat liver only a p prox. 50% of the 5 ' - n u c l e o t i d a s e is localised in this fraction [26]. T h e kinase r e s p o n s i b l e for the f o r m a t i o n of P t d l n s ( 4 , 5 ) P 2 is seen ( T a b l e II) to b e l o c a t e d a l m o s t exclusively in the p l a s m a m e m b r a n e fraction, where it is enriched 30-fold. The l y s o s o m e s
TABLE II DISTRIBUTION OF INOSITOL LIPID KINASES IN VARIOUS MEMBRANE FRACTIONS OF RAT LIVER Legend as in Table I. Abbreviations: RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum, n.d., not determined. Fraction
Spec. act. of Ptdlns kinase (pmo132p in Ptdlns4P/min per mg protein)
% Recovery of Ptdlns kinase activity
Spec. act. % Recovery of of Ptdlns4P Ptdlns4P kinase kinase (pmol 32p activity in Ptdlns(4,5) P2/min per mg protein)
Spec. act. of 5'-nucleotidase (#mol AMP hydrolysed/h per mg protein)
% Recovery 5'-nucleotidase
Homogenate RER SER
132, 94 71, 120 141, 228
(100) 1, 1 1.6, 1.2
11.5, 5.6 14, 14 28, 42
(100) 2.6, 2.1 3.8, 3.8
n.d. n.d. n.d.
n.d. n.d. n.d.
Homogenate Plasma membrane
79 + 14 (4) 2642 + 320 (6)
(100) 12, 18
32 + 11 (3) 954 + 250 (6)
(100) 9, 4
1.2 _+1.3 (6) 26.5 + 1.8 (5)
(100) 17 _+2.1
167
and the Golgi membranes contain little of the enzyme and that small activity seen in the Golgi could well be due to contamination by plasma membrane fragments. Fig. 3A shows the progress of labelling of Ptdlns4P and of Ptdlns(4,5)P 2 in plasma membranes in the presence of [32p]ATP. The rate of formation of Ptdlns4P is linear for only 2 min and declines slowly thereafter; the rate of formation of Ptdlns(4,5)P 2 is slow compared to that of Ptdlns4P. In all experiments there was an actual decline with time (over a 10 min period) in the amount of the two labelled species (data not shown). This was coincidental with the nearly total disappearance of ATP from the reaction mixture measured at 10 min (data not shown). Fig. 3B shows the rate of decline of ATP in the experiment shown in Fig. 3A. The activity of the ATPase in five separate experiments was 286 + 53 nmol A T P / m i n per mg protein at 30 °. It was therefore essential to keep the membrane addition to no more than 1 m g / m l in the reaction mixture, to add ATP to 2 mM instead of 1 mM and to use short incubation times. However, the breakdown of ATP produces another problem. Since the ATP is y-labelled, breakdown yields unlabelled ADP which in turn is converted to unlabelled ATP by
5
IO0
4
BO o~ c 60 c_
c
o. 3
the action of adenylate kinase (confirmed by HPLC). This means that the specific radioactivity of the ATP falls with time. In turn, the calculation of the formation of PtdIns4P, and more partaiculady that of PtdIns(4,5)P2, will be underestimated if the reaction is allowed to run for too long. Bearing this in mind, it is calculated that of the 40 nmol P t d I n s / m g protein in the plasma membrane fraction [19], some 7 nmol at the very least was converted to polyphosphoinositides. The method of Dorling and Le Page [20] used to prepare plasma membranes has present in the homogenising medium 0.5 mM C a 2+. Since this may activate a polyphosphoinositide phosphodiesterase [27], it may affect the phosphokinase assay in two ways: the generation of diacylglycerol may affect enzyme activity or the endogenous level of polyphosphoinositides would be depleted and thus alter the activity of inositol lipid kinases. To make sure that the presence of Ca 2÷ in the initial homogenising medium was not affecting the activity of either of the two inositol lipid kinases we prepared plasma membranes in the absence of C a 2+. This greatly reduces the yield but does not affect the qualitity of the preparation. We found that the activity of both the inositol lipid kinases (phosphatidylinositol kinase, 2628 p m o l / m i n per mg protein; phosphatidylinositol-4-phosphate kinase 1073 p m o l / m i n per mg protein) were similar to that shown in Table II for membranes prepared with Ca 2÷.
2
a. 2o ~ B E 0 c
0 0
3 Min
4
0
1
2
3 Min.
4
5
Fig. 3. A. Time-course of PtdIns4P and PtdIns(4,5)P 2 formation in rat liver plasma m e m b r a n e fraction. Assays were conducted as described in the legend of Fig. 2 except that the reactions were terminated by addition of 1 M perchloric acid. The precipitated protein was extracted for lipids as described in Fig. 2. The supernatants were retained for determination of ATP. Open circles, nmol of PtdIns4P formed per mg protein, and closed circles, nmol PtdIns(4,5)P 2 formed per mg protein in rat liver plasma membranes. B. Time-course of A T P hydrolysis by rat liver plasma m e m b r a n e fraction. Assays were performed on supernatants obtained after protein precipitation as described in A. supernatants were neutralised with K O H and the a m o u n t of A T P was determined by HPLC.
c_ o ci
(3. _L Q_
3-
2
1 E c
I
0
I
2
I
,4
I
6
I
~.
I 10
Min.
Fig. 4. Time-course of Ptdlns4P formation in rat hver lysosomes. Assay of phosphatidylinositol kinase was conducted as described in the legend to Fig. 2.
168
Endoplasmic reticulum
Mitochondria
As shown in Table I, microsomes of rat liver contain substantial amounts of both the phosphokinases under study, though at a low specific activity, and one may conclude that most of the activity is due to their content of Golgi and plasma membrane fragments. This is confirmed by the isolation of purified smooth and rough endoplasmic reticulum (Table II), which shows that activity is low compared to Golgi and plasma membrane.
The mitochondrial fraction showed negligible activity of the inositol lipid kinases, with specific activities well below those of the original homogenate (data not shown).
Lysosomes Collins and Wells [13] have reported that lysosomes of rat liver can phosphorylate phosphatidylinositol in the presence of ATP. Neither the enrichment nor the recovery of the enzyme activity in the lysosomal fraction was reported, however. Table III shows the results of our experiments with Triton-loaded lysosomes. The enrichment (29-fold) and the recovery (3%) of the kinase compares with 67-fold enrichment and 8% recovery of hexosaminidase and suggests that the kinase does indeed have a significant representation in lysosomes. Only the Golgi or plasma membrane fractions could contribute such large activities of the kinase, but marker enzymes for these (galactosyltransferase or 5'-nucleotidase and alkaline phosphodiesterase) show very tittle contamination by Golgi or plasma membrane fragments (data not shown). Negligible activity was detected for phosphatidylinositol-4-phosphate kinase (data not shown).
Cytosol Phosphatidylinositol-4-phosphate kinase activity has been identified as a cytosolic enzyme in brain [15]. To test the possibility that the cytosol may also contain this enzyme we prepared Golgi fraction (GF 1+2) and incubated this with cytosol in the presence of [32p]ATP. The presence of the cytosol had no effect on phosphatidylinositol kinase activity and on the level of Ptdlns4P formed and neither was any Ptdlns4P converted into Ptdlns(4,5)P 2, thus ruling out the presence of any cytosolic activity that was capable of converting the substrate presented by the membrane. Discussion In contrast to the endoplasmic reticulum, which is the principal location of phospholipid biosynthetic activity in rat liver [28], the synthesis of the polyphosphoinositides from Ptdlns occurs in a variety of cell-membranes. Downes and Michell [1] have reviewed the metabolism of the polyphosphoinositides and have discussed the data on the intracellular localisation of the kinases responsible for their synthesis from Ptdlns (see also Refs. 28 and 29 for reviews). Michell et al. [5] reported that phosphatidylinositol-4-phosphate kinase was con-
TABLE 1II LOCALISATION OF INOSITOL LIPID KINASES IN RAT LIVER LYSOSOMES Legend as in Table I. The specificactivityof phosphatidylinositol-4-phosphatekinase in the lysosomaifraction was negligible(5 pmol 32p in Ptdlns(4,5)P2/min per mg protein). Fractions
Homogenate Large granules Lysosomes
Spec. act. of Ptdlns kinase (pmol 32p in Ptdlns4P /rain per mg protein) 53+ 9 (4) 66+ 8(4) 1476+205(4)
% Recoveryof Ptdlns kinase activity
(100) 12.6+2.7 3 +1
Spec. act. of hexoseaminidase (# mol 2-nitrophenol formed/h per mg protein) 1.6+0.5 (6) 3.6+0.6(8) 105 +3.4(8)
% Recoveryof hexoseaminidase activity
(100) 30+7 8+2
169 centrated in the plasma membrane of rat liver. Phosphatidylinositol kinase has also been found in the Golgi apparatus [10] and in lysosomes [131. The present study confirms that the enzymes responsible for phosphorylation of Ptdlns are present in the plasma membranes, the Golgi apparatus and to some extent in the lysosomes of rat liver. We also report the distribution of the phosphatidylinositol-4-phosphate kinase. Because of the exclusive localisation of phosphatidylinositol-4phosphate kinase in the plasma membrane, it may be inferred that Ptdlns(4,5)P2 would be a reliable marker for the plasma membrane (providing that phospholipid exchange proteins do not transport it to other intracellular membranes). It may be inferred, too, that the breakdown of Ptdlns(4,5)P 2 in response to hormones must also be at the plasma membrane, generating diacylglycerol and inositol trisphosphate. Since the latter is released to the cytosol, it may be concluded that the lipid must be localised to the inner bilayer of the plasma membrane. It has been suggested that the phosphatidylinositol kinase is confined to Golgi membranes alone [10] but this is clearly not the case. The Golgi preparation is able to phosphorylate only 8% of its endogenous Ptdlns. This figure agrees with that found by Jergil and Sundler [10]. This degree of phosphorylation may reflect the amount of Ptdlns accessible to its enzyme. However, in the presence of Triton X-100, Jergil and Sundler [10] reported that this figure could be increased to only 20%. Thus it is very likely that product inhibition sets the limit as to how much Ptdlns can be phosphorylated in the Golgi, since in the plasma membrane, where Ptdlns4P is removed by further phosphorylation, more of the Ptdlns gets phosphorylated (see below). In the plasma membrane at least 17% of Ptdlns can be converted into polyphosphoinositides. The major contaminant of the plasma membrane preparation is the endoplasmic reticulum and it can be calculated from the specific activity of glucose6-phosphatase that it accounts for 20% of the preparation, as protein. Taking into account that endoplasmic reticulum has twice the amount of Ptdlns compared to plasma membrane [19], then the amount of Ptddlns that gets phosphorylated in the plasma membrane increases to 30%. As outlined in the Methods and Discussion the amount
of polyphosphoinositides formed in the plasma membrane will be underestimated so that the 30% is the absolute minimum. Thus the interesting possibility is raised that the major proportion of the inositol lipid in plasma membranes is present as the phosphorylated derivatives of Ptdlns. Such a situation does occur in the erythrocyte where the only membrane is a plasma membrane [31-33]. Phosphatidyhnositol kinase is known to be present in secretory granules of chromaffin cells [11], parotid [12] and the pancreatic fl-cell [8]. We presume that its presence in the Golgi apparatus of liver is analogous to these. However, the function of the enzyme in all four systems is obscure and may have some role in secretion, which is under hormonal control in chromaffin cells, the parotid and the pancreas, but not in the liver, which apparently produces and secretes the plasma proteins continuously. One possibility is that the phosphorylation of Ptdlns helps to provide a negative charge on the membrane which may be altered in the presence of Ca 2÷. Since Ca 2+ is known to inhibit the kinase [8], it is possible that the rise in cytosolic Ca 2÷ following cell activation observed in many cells may allow dephosphorylation to occur and the removal of the charge may allow membranes to come together before they fuse. The presence of Ptdlns kinase in lysosomes may play a similar role. The presence of both inositol lipid kinases in the plasma membrane is not surprising, since PtdIns(4,5)P 2 is implicated in the generation of two putative second messengers [2].
Acknowledgements We thank the Medical Research Council, The Wellcome Trust and the Vandervell Trust for grants in aid of our work.
References 1 Downes, C.P. and Micheli, R.H. (1982) Cell Calcium 3, 467-502 2 Berridge, M.J. (1984) Biochem.J. 220, 345-360 3 Michell, R.H. (1979) Trends Biochem. Sci. 4, 128-131 4 Hokin, L.E. and Hokin, M.R. (1964) Biochim. Biophys. Acta 84, 563-566 5 Michell,R.H., Harwood,T.L., Coleman, R. and Hawthorne, J.N. (1967) Biochim. Biophys.Acta 144, 649-658 6 Kai, M., White, G.L. and Hawthorne,J.N. (1966) Biochem. J. 101, 328-337
170 7 Rawyler, A.J., Roelofsen, B., Wirtz, K.W.A. and Op den Kamp, J.A.F. (1982) Febs Lett. 148, 140-144 8 Tooke, N.E., Hales, C.N. and Hutton, J.C. (1984) Biochem. J. 219, 471-480 9 Cooper, P.H. and Hawthorne, J.N. (1976) Biochem. J. 160, 97-105 10 Jergil, B. and Sundler, R. (1983) J. Biol. Chem. 258, 7968-7973 11 Phillips, J.H. (1973) Biochem. J. 136, 579-587 12 Oron, Y., Sharoni, Y., Lefkovitz, H. and Selinger, Z. (1978) in Cyclitols and Phosphoinositides (Wells, W.W. and Eisenberg, J.F., eds.), pp. 383-397, Academic Press, New York 13 Collins, C.A. and Wells, W.W. (1983) J. Biol. Chem. 258, 2130-2134 14 Hajra, A.K., Seguin, E.B. and Agranoff, B.W. (1968) J. Biol. Chem. 243, 1609-1616 15 Kai, M., Salway, J.G. and Hawthorne, J.N. (1976) Biochem. J. 106, 791-801 16 Glynn, I.M. and Chappell, J.B. (1964) Biochem. J. 90, 147-149 17 Cockcroft, S. (1984) Biochim. Biophys. Acta 795, 37-46 18 Bergeron, J.J.M. (1979) Biochim. Biophys. Acta 55, 493-503 19 Taylor, J.A., Limbrick, A.R., Allan, D. and Judah, J.D. (1984) Biochim. Biophys. Acta 769, 171-178 20 Dorling, P.R. and Le Page, R.N. (1973) Biochim. Biophys. Acta 318, 33-44
21 Gelter, D.M., Judah, J.D. and Nicholls, M.R. (1972) Biochem. J. 127, 865-874 22 Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S. and De Duve, C. (1968) J. Cell Biol. 37, 482-513 23 Butler, W.H. and Judah, J.D. (1969) J. Cell Biol. 44, 278-289 24 Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45-72 25 Farquhar, M.G., Bergeron, J.J.M. and Palade, G.E. (1974) J. Cell Biol. 60, 8-25 26 Stanley, K.K., Edwards, M.R. and Luzio, J.P. (1980) Biochem. J. 186, 59-69 27 Cockcroft, S., Baldwin, J.M. and Allan, D. (1984) Biochem. J. 221,477-482 28 Jelsema, C.L. and Morre, D.J. (1978) J. Biol. Chem. 253, 7960-7971 29 Michell, R.H. (1975) Biochim. Biophys. Acta 415, 81-147 30 Hawthorne, J.N. (1983) Biosci. Reps. 3, 887-904 31 Peterson, S.C. and Kirschner, L.B. (1970) Biochim. Biophys. Acta 202, 295-304 32 Schneider, R.P. and Kirschner, L.B. (1970) Biochim. Biophys. Acta 202, 283-294 33 Ferrell, J.E., Jr. and Huestis, W.H. (1984) J. Cell Biol. 98, 1992-1998