Hepatic Ins( 1,3,4,5)P4 3-Phosphatase Is Compartmentalized Inside Endoplasmic Reticulum*

In pursuit of the physiological role of inositol 1,3,4,5-tetrakisphosphate 3-phosphatase, which also attacks inositol pentakisphosphate and inositol hexakisphosphate with much higher affinity (Nogimori, K., Hughes, P.J., Glennon, M.C., Hodgson, M.E., Putney, J.W., Jr., and Shears, S.B. (1991) J. Biol. Chem. 266, 16499-16506), we have studied the subcellular distribution of the enzyme in liver. Initially, we had to overcome the problem that potent endogenous inhibitor(s) compromise the detection of this enzyme in vitro (Hodgson, M.E., and Shears, S.B. (1990) Biochem. J. 267, 831-834). We partially purified these inhibitor(s) by anion-exchange chromatography and gel filtration; inhibitory activity co-eluted with standard inositol hexakisphosphate and was depleted by treatment with phytase. Thus, subcellular fractions were pretreated with phytase before assay of 3-phosphatase activity. Our experiments revealed that the hepatic 3-phosphatase was nearly exclusively restricted to the endoplasmic reticulum, and there was little or no activity in either the cytosol, plasma membranes, mitochondria, or nuclei. Detergent treatment of microsomes indicated that there was 93 +/- 2% latency to mannose-6-phosphatase, an intraorganelle enzyme activity (Vanstapel, F., Pua, K., and Blanckaert, N. (1986) Eur. J. Biochem. 156, 73-77). Similar latencies were found for the hydrolysis of inositol 1,3,4,5-tetrakisphosphate (95 +/- 1%), inositol 1,3,4,5,6-pentakisphosphate (94 +/- 1%), and inositol hexakisphosphate (93 +/- 2%). Treatment of microsomes with either sodium carbonate or phosphatidylcholine-specific phospholipase C, to release luminal contents, led to solubilization of approximately 90% of 3-phosphatase activity. Thus, hepatic 3-phosphatase has a highly restricted access to inositol polyphosphates in vivo that needs to be accounted for in the determination of the physiological role of this enzyme.

It is now well established that agonist-stimulated phospholipase C activity results in accelerated breakdown of PtdIns(4,5)P2,' releasing Ins(1,4,5)P3 which mobilizes intra-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We (Nogimori et al., 1991) and others (Hoer and Oberdisse, 1991) have pointed out that the caveat to any proposals concerning 3-phosphatase activity in vivo is the possibility of differential subcellular compartmentalization of the enzyme in relation to its potential substrates. The lack of information on this topic is impeding our understanding of the physiological significance of this enzyme. It has been reported that phosphatase activity toward both Ins( 1,3,4,5,6)P5 and Ins(1,3,4,5)P4 may predominate on the surface of some cells (Carpenter et al., 1989), possibly inactivating putative activities of extracellular inositol phosphates (Vallejo et al., 1987;Perney and Kaczmarek 1992). However, there are no previous studies on the distribution of 3-phosphatase between the various subcellular fractions, other than a demonstration that the hepatic enzyme predominantly cosediments with a 100,000 x g particulate fraction (Nogimori et al., 1991).
Thus, it is with the aim of understanding the function of 3phosphatase that we have investigated its subcellular distribution in liver. However, we first had to accommodate the experimental problem that cells contain potent endogenous inhibitor(s) of this particular enzyme (Hodgson and Shears, 1990;Hughes and Shears, 1990). In this study we have found that the endogenous inhibitor(s) can be digested with phytase. By assaying 3-phosphatase activity in subcellular fractions pretreated with phytase, we have discovered that the enzyme 6161 is almost entirely restricted to the endoplasmic reticulum.
Moreover, using a variety of membrane-permeabilizing techniques, we discovered that the 3-phosphatase activity resides inside this organelle. This is the first report that an enzyme involved in inositol phosphate metabolism has restricted access to its potential substrates due to subcellular compartmentalization. Our work raises important questions concerning the physiological activity of this enzyme, but additionally our data have established the direction that future studies must take in order to resolve these problems.
Phytase activity was usually determined as described by Ullah and Gibson (1987) except that reaction mixtures were preincubated at 55 "C for 10 min prior to assay at 55 "C in a total volume of 300 pl.
In addition, all enzyme incubations were terminated with 33 p1 10% (w/v) SDS, prior to the measurement of Pi at 355 nm (Ullah and Gibson, 1987). One enzyme unit represents the activity that hydrolyzes 1 pmol of phytic acid in 1 min under the above experimental conditions. In some experiments, phytase activity toward [3H]InsP~ was monitored by high performance liquid chromatography (Nogimori et al., 1991).
Protein was assayed as described by Bradford (1976), using bovine serum albumin as a standard.
Preparation of Subcellular Fractions-Sprague-Dawley rats (150-200 g) were asphyxiated with CO,, and blood was removed from the livers by perfusion via the hepatic portal vein with buffer containing 250 mM sucrose, 10 mM Tris (pH 7.2 with HCl) plus a spectrum of protease inhibitors (Nogimori et al., 1991) except that phenylmethylsulfonyl fluoride was 1 mM. The following techniques were employed to isolate subcellular fractions from these livers. Plasma membranes were prepared as described by Bartles and Hubbard (1990). This method yields membrane sheet-like structures originating from si-nusoidal (basolateral) as well as bile canalicular (apical) surfaces of the hepatocyte. For the preparation of mitochondria, livers were homogenized by 8-10 up and down strokes of a Dounce-type loosefitting glass homogenizer (Wheaton, Melville, NJ; nominal clearance 0.0025-0.0035 inch) and the homogenate was processed according to Schneider and Hogeboom (1950). Microsomes were isolated as described by Vanstapel et al. (1986). Nuclei were isolated by the method of Blobel and Potter (1966). All the final preparations, with the exception of nuclei, were resuspended in 250 mM sucrose plus 10 mM Tris (pH 7.0 with HCl). Nuclei were resuspended in 50 mM Tris (pH 7.5 with HCI), 250 mM sucrose, 25 mM KC1, and 5 mM MgC12.
Purification of Insf1,3,4,5)P, 3-Phosphatase and Endogenous Inhibitorfsl-CHAPS-solubilized 100,000 X g particulate fractions (Nogimori et al., 1991), and 100,000 X g supernatants, were prepared from homogenates of either rat liver or brain. These were chromatographed on either a MonoQ HR 10/10 column (Hodgson and Shears, 1990) or a DEAE-Sepharose column (4.4 X 21 cm, Nogimori et al., 1991), using a gradient generated by mixing Buffer A (250 mM sucrose, 10 mM bis-Tris, 5 mM NaN3 (pH 7.4 with HC1) with Buffer B (Buffer A plus 500 mM NaCl). Aliquots of individual column fractions were heattreated by incubation at 90 "C for 15 min, followed by centrifugation for 5 min at 10,000 X g. The resultant supernatant was saved; it contained heat-stable endogenous inhibitor(s) (Hodgson and Shears, 1990). The 3-phosphatase activity eluted from the anion-exchange column was further purified as described by Nogimori et al. (1991).
For some experiments, a 4.5-ml aliquot of "heat-treated" endogenous inhibitor(s) was equilibrated with 1 X lo6 disintegrations/min [3H]InsP6 for 12 h at 4 "C. Then, 2.0 ml was applied at a flow rate of 0.2 ml/min to a Cellufine GCL-9OSF column (1.6 X 48 cm) equilibrated with 20 mM bis-Tris, 0.1 mM EDTA, 200 mM NaCl, 5 mM NaN3 (pH 7.4 with HCI). The column was eluted with the above buffer at a flow rate of 0.2 ml/min and the following fractions were collected fractions 1-6 (0-150 min) = 5 ml; fractions 7-95 (150-600 min) = 1 ml. The size exclusion column was calibrated under identical elution conditions using a Bio-Rad gel filtration standard kit with the addition of aprotinin (6.5 kDa). The elution of [3H]InsPG was identified by liquid scintillation spectrometry. The trace quantities of added [3H]In~Ps did not inhibit 3-phosphatase activity.
Purification of Phytase-Aspergillus ficuum phytase was purified from 100 mg of crude culture filtrate (Sigma catalog no. P9792; 4.1 Sigma units/ml). All procedures were conducted at 0-4 'C. The filtrate was resuspended in 100 ml of buffer containing 10 mM sodium acetate, 2 mM 2-mercaptoethanol, 5% (v/v) glycerol, pH 4.3 (with acetic acid), and incubated on ice for 30 min. The filtrate was centrifuged at 10,000 X g for 45 min, and the enzyme in the supernatant was purified using fast protein liquid chromatography and the procedures described by Ullah and Gibson (1987), i.e. SP-Trisacryl M (16 X 130 mm; IBF Biotechnics (Sepracor Inc.)), followed by DEAE Trisacryl M (10 X 140 mm; IBF Biotechnics (Sepracor Inc.)). At this step all the phytase activity eluted as a single peak of activity at approximately 50 mM KC1 (designated "Phytase 11" by Ullah and Gibson, 1987). The final purification step used a MonoP HR 5/20 chromatofocusing column. Phytase activity coeluted with the second major protein peak at pH 4.5-5.0. The three peak fractions of enzyme activity were pooled and stored as 200-fl1 aliquots in the absence of glycerol at -20 "C until use.
Removal of Endogenous Inhibitorfs) of 3-Phosphatase in Tissue Extracts by Phytase-Phytase treatment of either DEAE-purified endogenous inhibitor(s) (prepared as described tiy Fig. l), liver homogenates, or subcellular fractions (1-3 mg protein/ml) was by incubation of tissue with 0.01 units/ml phytase for 60 min at 37 "C in 3-phosphatase assay medium (except that CHAPS was omitted and 250 mM sucrose was added). The homogenates or subcellular fractions were then centrifuged at 100,000 X g for 15 min at 4 "C (Beckman TL-ultracentrifuge, TLA 100.3 rotor). Pellets were resuspended in 1 ml of buffer containing 10 mM Tris (pH 7.0 with HCl), 250 mM sucrose, and these were washed twice by recentrifugation. Finally, pellets were resuspended and diluted appropriately in the buffer for assay of 3-phosphatase activity. These final tissue preparations, when incubated in the absence of detergents, exhibited no residual phytase activity, even in incubations of up to 2 h (data not shown). The effectiveness of the removal of inhibitors of 3-phosphatase was confirmed in two ways. First, internal standards of [3H]InsP6 were found to be completely hydrolyzed to [3H]InsP. Second, the phytase-treated tissue extracts, after themselves being heat-treated, did not inhibit purified 3-phosphatase.
Perturbation of Microsomal Integrity-For detergent treatment, microsomes (2 mg protein/ml) were resuspended as described above and incubated on ice with the addition of a range of concentrations of either CHAPS (0-4 mM for 60 min) or Triton X-100 (0-2 mM for 30 min).
NazCO, treatment was performed by incubation of microsomes (0.5-1 mg protein/ml) in 1 ml of 100 mM Na2C03 (pH 11.4) on ice for 30 min (Fujiki et al., 1982). The pH was then adjusted to 7.0 with an appropriate volume of 400 mM MES. Phospholipase C treatment of 0.5-1 mg microsomal protein/ml was performed as described by Urade et al. (1992) except that their buffer was replaced with 1 ml of 250 mM sucrose, 10 mM Tris (pH 7.0 with HCI). Following these two treatments, samples were centrifuged (100,000 X g, 20 min, 4 "C). Both the supernatants and the pellets were saved and then assayed for mannose-6-phosphatase and Ins(1,3,4,5)P4 3-phosphatase as described above.
Panel B shows an experiment in which the inhibitory activity in 12 X 20-ml fractions eluted from a DEAE column were pooled and equilibrated with [3H] InsP, for 12 h at 4 "C (see "Experimental Procedures"). Then, 2 ml of this mixture was applied to a Cellufine GCL-90 sizeexclusion column as described under "Experimental Procedures." Endogenous inhibitory activity (0) was assayed as described above, except that [5-3zP] Ins(1,3,4,5)P4 was used as substrate in  (Fig. L 4 ) or a DEAE-sepharose column (data not shown). The resultant fractions were heat-treated (see "Experimental Procedures") and added to incubations containing purified 3-phosphatase. A broad peak of inhibitory activity toward this enzyme was clearly demonstrated and, moreover, was separated from the peak of 3-phosphatase itself (Fig. lA). From the enzyme activities that were expressed in the soluble and particulate fractions after anion-exchange chromatography, we determined that 94 & 1% ( n = 2) of total hepatic Ins(1,3,4,5)P4 3phosphatase activity was originally associated with the particulate fraction (data not shown). In contrast, it was the soluble fraction that contained the bulk (approximately 90%) of total heat-stable inhibitory activity toward 3-phosphatase. This was established by incubating aliquots of heat-treated soluble and particulate fractions with purified 3-phosphatase (data not shown). Nevertheless, the minor proportion of inhibitory activity that associates with hepatic membranes is still sufficient to substantially inhibit the 3-phosphatase activity that is also present in this fraction of the cell (see above). Therefore, in order to study the distribution phosphatase between the various hepatic organelles, we had to develop a routine means of eliminating these membraneassociated inhibitor(s). We (Hughes and Nogimori et al., 1991) have determined that InsP, is a potent inhibitor of 3-phosphatase activity (Ki = 2 nM). Thus it is possible that InsPs makes a major contribution to the endogenous inhibition of the enzyme. Consistent with this proposal, we have found that upon gel filtration, the particulate inhibitory activity coeluted precisely with an internal standard of [3H]InsP6 (Fig. 1B). Note that the apparent molecular mass of InsPs (9 kDa, Fig. 1B) was unexpectedly high. This is probably in part due to the heavily hydrated nature of the InsP, salt under physiological conditions (Blank et al., 1971).
We considered that if InsPs, and/or a closely related polyphosphate, was responsible for endogenous inhibition of 3phosphatase, then it should be possible to eliminate such inhibition by treatment with phytase. To test this possibility, inhibitor(s) purified from the CHAPS-solubilized particulate fraction (see "Experimental Procedures") were incubated with sufficient phytase that internal standards of 1 mM [3H]InsPs were completely hydrolyzed to [3H]InsP. Phytase was then itself inactivated by heat treatment (Table I). Heat treatment alone did not affect the activity of the inhibitor (Table I). However, sequential treatment of the inhibitor, first with phytase and then with heat, completely eliminated its ability to inhibit Ins(1,3,4,5)P4 3-phosphatase (Table I). Thus, we next investigated the intracellular distribution of 3-phosphatase in subcellular fractions treated with phytase (see below).

Ins(1,3,4,5)P4 3-Phosphatase Activity in Homogenates and Subcellular Fractions Which Were Depleted of Endogenous
Inhibitor(s)-In incubations containing liver homogenates (2.6-4.6 mg protein/ml) the apparent Ins(1,3,4,5)P4 3-phosphatase activity was 4.8 f 0.6 pmol/min/mg protein (mean f S.E., n = 10). After separation of the particulate fraction from the homogenates by centrifugation at 100,000 x g, the apparent 3-phosphatase activity increased 12-fold (to 57 f 8 pmol/ min/mg protein, n = 10). This reflects the removal of those endogenous inhibitor(s) that are released into the soluble fraction of liver homogenates (see above, and Hodgson and Shears, 1990). Nevertheless, substantial inhibitory activity persists in the 100,000 X g particulate fraction (see above). When homogenates were pretreated with phytase to digest endogenous inhibitor(s) (see "Experimental Procedures") and then the particulate fraction was isolated, the apparent 3phosphatase activity associated with these membranes was

Effect of phytase upon endogemus inhibitor(s) of purified Ins(l,3,4,5)P4-3-phosphatose
Purified 3-phosphatase was incubated in triplicate with 5 pM ['HI Ins(1,3,4,5)P4 as described under "Experimental Procedures." Where indicated (lines 2-4) incubations also contained a 1:lO dilution of heat-treated endogenous inhibitor purified on a DEAE column as described in the legend to Fig. 1. In some incubations the inhibitor was pretreated by either (line 3) heating at 100 "C for 1 h, or (line 4) phytase (see "Experimental Procedures") followed by heat (100 "C, 1 h). In control experiments (not shown) phytase did not metabolize Ins(1,3,4,5)P4 after heat treatment (100 "C, 1 h). Data are means f standard errors from triplicate incubations. One additional experiment gave similar results.  -fold greater (148 f 21 pmol/min/mg protein, n = 10) than that originally expressed in homogenates. The latter was therefore the appropriate value to refer to when estimating the enrichment of 3-phosphatase in subcellular fractions. Table I1 describes the -fold enrichment of selected markers in our preparations of subcellular fractions. Markers for plasma membranes (alkaline phosphodiesterase), microsomes (arylesterase), mitochondria (glutamate dehydrogenase), and nuclear fractions (DNA) were enriched 25-, 5-, 3-, and 16fold, respectively, which are all typical of subcellular preparations from liver (Ali et al., 1990;Schneider and Hogeboom, 1950;Joseph and Williams, 1985;Widnell and Tata, 1964). Each of these fractions were treated with phytase, which was then itself removed by two cycles of washing and centrifugation (see "Experimental Procedures"). Importantly, aliquots of all the phytase-treated subcellular fractions were, after heat treatment, found not to inhibit purified 3-phosphatase in incubations containing CHAPS (data not shown). This result indicates the absence of any residual endogenous inhibitory activity, including any that might have been intravesicular and escaped attack by phytase which out of necessity was added to subcellular preparations in the absence of detergent.
Among the various subcellular fractions, the microsomes were the only organelles to contain an enrichment of 3phosphatase (approximately 4.5-fold) that quantitatively matched the enrichment of the appropriate specific marker (Table 11). Furthermore, the percentage yield in the microsomal preparations of total cellular 3-phosphatase and arylesterase were very similar: 15 f 2% and 17 f 2%, respectively. Finally, the relatively small amounts of 3-phosphatase activities that were found in other subcellular fractions were closely matched by contamination of the marker for endoplasmic reticulum, with the possible exception of a small amount of 3-phosphatase activity in plasma membranes. Note also that the contaminating 3-phosphatase activities, as well as that in microsomes, were all increased by between 1.5-and 3-fold by phytase treatment (Legend to Table I), suggesting that endogenous inhibitors were somewhat uniformly distributed throughout the different particulate fractions of the cell.

,5)P4 3-Phosphatase in
Microsomes-We compared the apparent microsomal 3-phosphatase activity in intact vesicles with the activity upon permeabilization by CHAPS. Mannose-6-phosphatase activity was the control marker for the latency of our microsomal preparations (Vanstapel et al., 1986), i.e. 93% (Table 111); presumably about 7% of our microsomal vesicles were inherently leaky. Unexpectedly, Ins( 1,3,4,5)P4 3-phosphatase was also latent to the same extent (Table 111). We considered the possibility that the apparent latency of 3-phosphatase might be alternately explained by a nonspecific detergent-induced activation of the enzyme. However, this is extremely unlikely, since over a range of concentrations of two different types of detergent (CHAPS, which is zwitterionic, and Triton X-100, which is non-ionic), the curves that describe the progressive increases in activities of 3-phosphatase and mannose-6-phosphatase (Fig. 2) are virtually superimposable. The remote possibility that 3-phosphatase and mannose-6-phosphatase were the same enzyme was excluded since purified 3-phosphatase did not hydrolyze mannose-6-phosphate (data not shown). Thus, Ins(1,3,4,5)P4 3-phosphatase is apparently compartmentalized inside endoplasmic reticulum. This enzyme also attacks Ins(1,3,4,5,6)P~ and InsPs (Nogimori et al., 1991). We found that these alternative substrates were also not significantly hydrolyzed by microsomal vesicles until these membranes were disrupted with detergent (Table 111).

,5)P4-3-phosphatase and various markers in subcellular fraction
Details of the preparation of subcellular fractions, and the enzyme assays, are given under "Experimental Procedures"; 4 mM CHAPS was included in all assay media. Data are means t standard errors, followed in parentheses by the number of separate preparations of subcellular fractions. The -fold enrichment of subcellular markers relate to assays performed on homogenates, except for the 3-phosphatase, where enrichment in phytase-treated subcellular fractions was compared with activity in a phytase-treated 100,000 X g particulate fraction (see under "Results" and "Experimental Procedures"). The apparent 3-phosphatase activities in subcellular fractions increased following phytase treatment by the following -fold values: 3.2 f 0.3 (plasma membranes), 3.4 f 0.8 (mitochondria), 3.2 & 1.7 (nuclei), or 1.4 f 0.1 (microsomes).
Marker enrichment relative to the bulk particulate fraction (-fold) mM phosphocreatine, 50 p~ GTP, 5 mM K' succinate, 10 pg/ ml creatine kinase). Enzyme latency was also unaffected (data not shown) by the further addition to this medium of Ca2+/ BAPTA buffers to set the free [Ca"] to either 0.1 p~, 0.5 p~ or 1 p~ in the presence of 10 p~ calmodulin, or by 30 min pretreatment of microsomes by 2.5 units/ml protein kinase C, or by 200 p~ A1C13, which in some circumstances may activate 3-phosphatase (Loomis-Husselbee et al., 1991). We further investigated the location of 3-phosphatase in microsomes by two additional methods that do not use detergents, i.e. treatment with either Na2C03 (pH 11.4;Fujiki et al., 1982) or phosphatidylcholine-specific phospholipase C (Urade et al., 1992). These methods have the further advantage that they have been used to specifically release proteins that are either free in the microsomal lumen, and possibly those that are loosely associated with the luminal face of the membrane. Following such procedures (see "Experimental Procedures" for details) the disrupted microsomes were centrifuged at 100,000 x g, and both the resultant soluble and particulate fractions were separately assayed for 3-phosphatase. In both cases over 90% of total 3-phosphatase was recovered in the soluble fraction (Table IV). As a control, we also investigated the distribution of membrane-bound mannose-6-phosphatase in the disrupted microsomes. Unfortunately, phospholipase C treatment totally inactivated mannose-6-phosphatase activity (Table IV). While the Na2C03 treatment reduced mannose-6-phosphatase activity by 45%, the activity that remained was all associated with the partic- Both mannose-6-phosphatase (A) and 3-phosphatase (0) activities were assayed (see "Experimental Procedures") at 20 "C in 50 mM Tris, 250 mM sucrose, 2 mM EDTA (pH 6.5 with HCl). Maximum activities obtained in the presence of detergents were taken as 100%. Data shown from a single experiment are typical of three experiments performed on separate preparations.  Ins(1,3,4,5)P,-3-phosphatase and mannose-6phosphatase between a 100,000 X g supernatant and pelkt prepared from microsomes disrupted with either Na2C03 or phospholipase C Microsomes were treated with either Na2C03 or phosphatidylcholine-specific phospholipase C and then the disrupted vesicles were separated into a 100,000 X g supernatant and pellet (see "Experimental Procedures"). Data are means rf: S.E. from the number of preparations indicated in parentheses. ND = not detected. Note that Na2C03 and 100% by phospholipase C. mannose-6-phosphatase was inactivated 45% by treatment with  (Table IV).

DISCUSSION
Some workers have suggested that Ins(1,3,4,5)P4 3-phosphatase activity represents a physiological means of sustaining Ins(1,4,5)P3 levels during cell stimulation . More recently, we have shown that Ins(1,3,4,5,6)P5, and particularly InsP6, are both higher affinity substrates of this enzyme (Nogimori et al., 1991). These observations led us to propose that the higher polyphosphates were more likely to be the physiologically relevant substrates. However, Oberdime and co-workers (Hoer et al., 1991), and our laboratory (Nogimori et al., 1991), have emphasized that we are unlikely to understand the physiological activity of this enzyme in vivo until we determine where in the cell the enzyme and its substrates are located.
It is against this background that we can now add important information on the subcellular location of the hepatic 3phosphatase, which we obtained by using phytase to eliminate endogenous inhibitory activity from subcellular fractions.' Our experiments have revealed that the 3-phosphatase resides inside the endoplasmic reticulum, without apparent access to its inositol phosphate substrates. We have incubated microsomes under a variety of conditions (see "Results"), in an effort to exclude the possibility that one or more inositol phosphates might be transported into endoplasmic reticulum in uiuo by a mechanism that might not be revealed by our in uitro experiments. We were still unable to obtain any evidence that Ins(1,3,4,5)P4 is normally metabolized by this enzyme. These data are of substantial physiological interest. They are particularly strong evidence that the dephosphorylation of While phytase undoubtedly hydrolyzed membrane-associated InsP6, this enzyme will also have removed any other inositol polyphosphates (and conceivably other unsuspected polyphosphates) that might contribute to endogenous inhibition of 3-phosphatase.
Ins(1,3,4,5)P4 to Ins(1,4,5)P3 does not contribute to the regulation of Ca'+ mobilization in intact liver cells. An important practical application of this discovery relates to the potential for using hepatocytes, and other intact cells in which 3phosphatase might be similarly compartmentalized, for studies into the effects upon Ca*+ signaling of microinjection of Ins(1,3,4,5)P4. Irvine (1992) has emphasized that a major problem with such an approach lies in the interpretation of positive effects of Ins(1,3,4,5)P4 upon Ca2+ mobilization, in view of the possibility that such phenomena arise from generation of Ins(1,4,5)P3 by the 3-phosphatase. Our results make this a much less likely scenario.
Our data also indicate that the alternative substrates of the 3-phosphatase, Ins(1,3,4,5,6)P5 and Imp6, also have a very restricted access to this enzyme in vivo. These results provide an explanation for the otherwise paradoxical observation that intact mammalian cells usually contain only very low steadystate levels of both Ins(1,4,5,6)P4 (Stephens et al., 1988a;Balla et al., 1989;McConnell et al., 1990;Wong et al., 1992) and Ins(1,2,4,5,6)P5 (Menniti et al., 1990;Mattingly et al., 1991;Nogimori et al., 1991;Wong et al., 1992), despite their immediate metabolic precursors being high affinity substrates of the 3-phosphatase in uitro (Nogimori et al., 1991). In liver, only 5-7% of total cellular activity was associated with 100,000 x g soluble fractions (see "Res u l t~" ) .~ Yet even this small value probably overestimates the proportion that might be present in liver cytosol in vivo. We have obtained evidence that the enzyme is either soluble inside the lumen of the endoplasmic reticulum or at least loosely bound to the inner membrane face (Table IV). Upon cell homogenization, which fragments the endoplasmic reticulum into microsomes, there is an inevitable loss to the soluble fraction of a proportion of loosely associated luminal contents (Beckers etal., 1987). Thus, none of the hepatic 3-phosphatase may be genuinely cytosolic. In this event, perhaps when small amounts of Ins(1,4,5,6)P4 and Ins(1,2,4,5,6)P5 are observed in cell preparations, they are actually only derived from a minor proportion of cells that have begun to lose some structural integrity. It will therefore be of physiological interest to understand why levels of either Ins(1,4,5,6)P4 or Ins(1,2,4,5,6)P5 are atypically high in certain transformed cells, such as the AR4-2J pancreatoma (Menniti et al., 1990), src-transformed rat fibroblasts (Mattingly et al., 1991), Epstein-Barr virus-transformed human B-lymphocytes (Mc-Connell et al., 1991), and Jurkat T-lymphocytes (Guse and Emmrich, 1991). Perhaps transformation has perturbed the barrier that normally separates the 3-phosphatase from its substrates. Alternately, the subcellular location of 3-phosphatase, and hence its function, may be different in some nonhepatic tissues. In this respect, the Ins( 1,3,4,5)P4 3-phosphatase in human erythrocytes is an integral protein on the inner face of the plasma membrane (Estrada-Garcia et al., 1991). Our marker enzyme data do not entirely exclude the possibility that a very minor proportion of 3-phosphatase might be associated with hepatic plasma membranes (Table 11). Nevertheless, the erythrocyte, being an atypical, highly differentiated cell type with a dearth of intracellular membranes, is an inappropriate model for predicting the intracellular distri-Even in rat brain, where previous attempts to understand the physiological activity of the 3-phosphatase were based upon studies of apparently cytosolic activity (Hoer et al., 1988Hoer and Oberdisse, 1991), we have now found that 90% of total cellular 3phosphatase activity actually cosediments with a 100,000 X g particulate fraction. As in our current experiments with rat liver, this observation was made by comparing 3-phosphatase activities in ~~~,~O O x g supernatants and CHAPS-solubilized 100,000 X g particulate fractions, after ion-exchange chromatography. bution of 3-phosphatase in most other cell types. The bulk of Ins(1,3,4,5)P4/Ins(l,3,4,5,6)P5 phosphatase activity has also been reported to reside on the surface of NTH-3T3 fibroblasts (Carpenter et al., 1989) where it has been suggested to inactivate putative extracellular signaling activities of inositol phosphates (Perney and Kaczmarek, 1992). However, the specificity of this putative extracellular enzyme has not been determined.
The conclusion that hepatic 3-phosphatase does not make a large contribution to inositol phosphate turnover in vivo should lead us to consider that this enzyme may perform other functions. Although our studies do not directly address this issue, our identification of the intracellular location of this protein does represent important groundwork that should form the basis of further investigations into this topic. For example, luminal enzymes of the endoplasmic reticulum are frequently either exported from the cell in a secretory pathway or they participate in the processing of such secreted proteins (for review, see Pelham, 1989). Future research into the function of the 3-phosphatase should be directed at possible contributions to this, and probably other, physiological processes that may not even be directly related to the turnover of inositol polyphosphates.