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Letter to the editor: Squalene is Localized to the Plasma Membrane in Bovine Retinal Rod Outer Segments
Exp. Eye Res. (1997) 64, 279–282
LETTER TO THE EDITORS
Squalene is Localized to the Plasma Membrane in Bovine Retinal Rod Outer Segments Squalene, an isoprenoid hydrocarbon, is an obligatory, but transient, intermediate in the de novo synthesis of sterols in all eukaryotic cells (Schroepfer, 1981). In most cells and tissues (with the notable exception of liver), squalene represents an extremely minor percentage of the total lipids. We previously reported the unusual finding that the retina of the leopard frog (Rana pipiens), an animal commonly used in vision research, contains appreciable amounts of squalene, particularly in the rod outer segment (ROS) membranes (Keller, Fliesler and Nellis, 1988 ; Keller and Fliesler, 1990). The cholesterol}squalene mole ratio in frog ROS membranes is approximately 10 ; since cholesterol accounts for E 10 mol % of the total ROS membrane lipid, squalene represents E 1 mol %. Squalene is also present in bovine ROS membranes, but at levels approximately 100-fold lower than in the frog ROS (see below). Certain lipids have been shown to exhibit a preferential localization to the plasma membrane of the ROS, compared to the disc membranes. For example, cholesterol is enriched E 4-fold (normalized to total phospholipid content) in the plasma membrane relative to the majority of the mature disc membranes, and it also is enriched E 6-fold in the basal disc membranes relative to the older discs located toward the distal tip of the ROS (Boesze-Battaglia and Albert, 1989 ; Boesze-Battaglia, Hennessey and Albert, 1989 ; Boesze-Battaglia, Fliesler and Albert, 1990). Similarly, the ROS plasma membrane is relatively enriched (compared to the disc membranes) in the fatty acids C14 : 0 (E 14-fold), C18 : 2 (E 8-fold), and C18 : 3 (E 7-fold), whereas the mature discs are relatively enriched (compared to the plasma membrane) in C22 : 6 (E 7-fold) (Boesze-Battaglia and Albert, 1989). Since the relative distribution of squalene in these two distinct ROS membrane compartments is not known, the distribution of squalene, relative to cholesterol, was examined in highly purified bovine ROS plasma membranes and disc membranes. The results found suggest that most of the squalene in the bovine ROS is localized to the plasma membrane, while mature disc membranes are virtually devoid of squalene. Frozen, dark-adapted bovine retinas (50 retinas per preparation ; J. Lawson, Inc., Lincoln, NE, U.S.A.) were used for the preparation of purified ROS membranes (disc membranes and plasma membrane), using modifications of the method of Molday and Molday (1987), as described in detail previously (BoeszeBattaglia and Albert, 1989 ; Boesze-Battaglia, 0014-4835}97}02027904 $25.00}0}ey960201
Hennessey and Albert, 1989). Based upon both lipid phosphorus determination [using a modification of the method of Bartlett (1959), as described by Litman (1973)] and protein assay (micro-BCA method, per Pierce Chemical Co., Rockford, IL, U.S.A.), the recovered amount of ROS plasma membrane was 2–3 % that of the recovered disc membrane fraction. Aliquots of each membrane fraction (ca. 0±01 % of the disc membranes and ca. 10 % of the plasma membranes) were taken for SDS-PAGE analysis to confirm the characteristic protein pattern of each membrane fraction (Molday and Molday, 1987). Approximately 1 % of the disc membrane fraction and ca. 75 % of the plasma membrane fraction from each of three independent ROS membrane preparations were then subjected to saponification, extraction of the nonsaponifiable lipids with petroleum ether, and analysis of the nonsaponifiable lipids by reverse-phase HPLC, as described in detail elsewhere (Keller, Fliesler and Nellis, 1988 ; Keller and Fliesler, 1990). Serial dilutions of authentic cholesterol and squalene (Sigma Chemical Co., St. Louis, MO, U.S.A.) were used to calibrate the integrated detector response factors for each lipid (using detection at 210 nm), which permitted quantitation of the levels of those lipids in the biological samples analysed. Under the conditions employed, we could detect & 1 pmol of cholesterol (& 386 pg) and & 0±1 pmol of squalene (& 4±11 pg). The difference in detection sensitivity is due to the ten-fold greater extinction coefficient of squalene relative to that of cholesterol. Representative reverse-phase HPLC chromatograms of nonsaponifiable lipids from the disc membrane and plasma membrane fractions are shown in Fig. 1. The elution positions of cholesterol (designated ‘ C ’ ; retention time 12±0³0±1 min) and squalene (designated ‘ S ’ ; retention time 14±0³0±1 min) are indicated. Under the conditions employed, the cholesterol peak in the disc membrane fraction (panel A, 89 nmol) is nearly at full scale, whereas that corresponding to squalene is barely detectable by visual inspection (but is quantifiable by peak integration as 64 pmol). In this particular preparation, the cholesterol}squalene mole ratio was 1±4¬10$ ; the value (mean³..) obtained for all three preparations was 1±7³0±7¬10$. In striking contrast, the chromatogram obtained for the ROS plasma membranes (panel B ; using a sensitivity 16 times greater than in panel A) exhibits a peak corresponding to squalene (10 pmol) that is greater in height than the peak corresponding to cholesterol (55 # 1997 Academic Press Limited
280
LETTER TO THE EDITORS (A)
(B)
6.72
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F. 1. Reverse-phase HPLC chromatograms of nonsaponifiable lipids isolated from (A) ROS disc membranes and (B) ROS plasma membrane. The elution positions of cholesterol (C) and squalene (S) are indicated. Note that the detector sensitivity employed for obtaining the data in panel (B) was 16-fold greater than that employed to obtain the data shown in panel (A) (relative response, absorbance at 210 nm).
pmol). The mole ratio of cholesterol}squalene in this plasma membrane preparation was 5±5 ; for the three preparations analysed, this value was 6±3³2±9. Thus, when one normalizes the squalene content relative to that of cholesterol (mol : mol), the ROS plasma membrane fraction exhibits nearly a 300-fold enrichment in squalene compared to the bulk of the mature disc membranes. Using these empirically determined ratios and the values previously determined for the cholesterol} phospholipid mole ratio of the bovine ROS plasma membrane (0±38) and the average disc membrane population (0±11) (Boesze-Battaglia, Hennessey and Albert, 1989), the calculated average values for the squalene}phospholipid mole ratio of the ROS plasma membrane and disc membranes are 6±1¬10−# and 6±5¬10−&, respectively. This would suggest almost a 10$-fold concentration difference in squalene between the plasma membrane and mature disc membranes. Since the disc membranes account for about 15 times more of the total ROS membrane mass than does the plasma membrane, the preferential localization of squalene to the plasma membrane, relative to the discs, is on the order of 70-fold. Comparing this value with the compositional differences reported for ROS plasma membrane and disc membranes with respect to cholesterol and various phospholipid acyl chains, the squalene concentration difference is the largest observed for any lipid species in ROS membranes (i.e., on the order of 10-fold greater than other lipid differences).
Although bovine ROS are clearly squalene-deficient compared with frog ROS, we employed bovine ROS in this study because the methods available for separating and purifying the plasma membrane and disc membrane fractions to near homogeneity have been established for bovine, but not frog, retina. Due to the relatively large tissue mass, one can obtain sufficient yields of ROS plasma membrane from bovine retinas to perform detailed biochemical analyses. In contrast, our repeated attempts to obtain a similar degree of fractionation and purification of ROS plasma membranes from frog retina (typically using 200–250 retinas per preparation), and in sufficient yields for similar biochemical analyses, have been unsuccessful. Hence, although considerably more is known about isoprenoid lipid metabolism in the frog retina than in any other species, and although the frog ROS exhibits a profound enrichment in squalene, we have not been able to determine the relative distribution of squalene in the membranes of the frog ROS. The squalene found in ROS membranes most likely arises from de novo synthesis in the rod cells. The formation of squalene in the retina from common, radiolabeled de novo precursors (e.g., [$H]acetate, [$H]mevalonate) has been demonstrated both in vitro and in vivo in frog and cow eyes (Fliesler, 1979 ; Fliesler and Schroepfer, 1986 ; Keller, Fliesler and Nellis, 1988 ; Keller and Fliesler, 1990 ; Fliesler, Florman and Keller, 1995). In all those studies, radiolabeled squalene, rather than cholesterol, was by far the predominant nonsaponifiable lipid product formed. In the frog retina, a substantial portion of the total radiolabeled squalene formed is subsequently transported to and incorporated into the ROS membranes, where it turns over independent of and without conversion to cholesterol (Keller and Fliesler, 1990 ; Fliesler, Florman and Keller, 1995). The incorporation of newly synthesized squalene into the ROS has been observed also in the bovine retina in vitro (Florman et al., 1994). As discussed in detail previously (see Fliesler, Florman, and Keller, 1995), the retinal pigment epithelium is not likely to contribute significantly to the squalene mass in the ROS. The presence of appreciable steady-state amounts of squalene, and its preferential segregation in the plasma membrane vs. the disc membranes of the ROS, begs at least two questions : (1) What is the biological role of squalene in the ROS ? and (2) What are the driving forces that promote the relative segregation of squalene into the plasma membrane ? In regard to the first question, prior studies on squalene metabolism in the frog retina would suggest that it is not serving its classic biogenic role as a cholesterol precursor, since squalene is not converted to cholesterol in the ROS (Fliesler, Florman and Keller, 1995). Clues to the potential role of squalene in the ROS may be derived from studies concerning its biophysical effects in artificial membranes. In this regard, Lohner and coworkers (Lohner
LETTER TO THE EDITORS
et al., 1993) have shown that squalene, at levels & 6 mol % of the total lipid, can destabilize the bilayer (‘ lamellar ’ phase) structure of artificial phospholipid membranes, thereby promoting the formation of nonbilayer structures, e.g., the ‘ inverse hexagonal ’ (HII) phase. Such alternate phase structures have been shown to have profound effects on the properties and functions of biological membranes, including the translational and rotational mobility of membrane proteins, the activity of membrane enzymes, and ion permeability (reviewed in Cullis and Hope, 1985 ; Gruner, 1992 ; Glaser, 1993). Our calculations indicate that the average squalene content of the bovine ROS plasma membrane approaches 6 mol % of the total lipid. While this concentration of squalene may be barely sufficient to perturb membrane structure (assuming isotropic distribution), nonhomogeneous segregation of squalene could provide localized concentrations of squalene sufficient to promote non-bilayer phases in discrete membrane domains, without altering the bulk, longrange bilayer structure. One potential consequence of such localized alternate membrane phase structure is the promotion of membrane fusion events (reviewed in Bentz and Ellens, 1988 ; Yeagle, 1993), which are apparently requisite in the processes of disc morphogenesis and disc shedding (reviewed by Besharse, 1986). Recently, it has been shown that the ability of ROS disc membranes to undergo fusion is correlated with the presence of non-bilayer, ‘ isotropic ’ phases, which can be promoted by increasing the molar concentration of retinol or retinal in the membrane (Boesze-Battaglia and Yeagle, 1992 ; Boesze-Battaglia et al., 1993). Hence, the distribution and concentration of free isoprenoids and}or retinoids might be important in controlling the spatial and temporal frequency of membrane fusion events involved in ROS membrane renewal and turnover. With regard to the second question, the answer is not readily apparent. Although squalene tends to partition preferentially into ‘ liquid-crystalline ’, as opposed to ‘ gel ’, phases in aqueous phospholipid dispersions (Lohner et al., 1993), both the disc membranes and plasma membrane are likely to exist in the liquid-crystalline state at physiological temperatures (see Cullis and Hope, 1985). In contrast to cholesterol, which can exchange between physically discontinuous membranes across an aqueous space (House, Badgett and Albert, 1989) and apparently does so as the disc membranes are displaced progressively toward the distal tip of the ROS (BoeszeBattaglia, Hennessey and Albert, 1989 ; BoeszeBattaglia, Fliesler, and Albert, 1990), squalene (a hydrocarbon) would not be expected to partition between membranes via an aqueous environment. Hence, the ‘ molecular segregation ’ of squalene must occur during or immediately prior to the maturation of new discs near the base of the ROS. A similar process is likely involved in the absolute sorting of various
281
membrane proteins to either the plasma membrane (e.g., the GLUT-1 glucose transporter, the cGMP-gated channel and channel-associated protein, the Na+}K+}Ca#+ exchanger) or the disc membranes (e.g., ROM-1, peripherin}rds, ‘ rim protein ’) (see Molday and Molday, 1987, 1993). However, the details of such sorting mechanisms remain to be elucidated. Acknowledgements This study was supported by U.S.P.H.S. (NEI}NIH) grants EY07361 (SJF and RKK), EY10420 (KB-B), and EY03328 (ADA), and by an unrestricted departmental grant from Research to Prevent Blindness, Inc. (SJF).
STEVEN J. FLIESLERa* KATHLEEN BOESZE-BATTAGLIAb ZOPHIA PAWc R. KENNEDY KELLERd ARLENE D. ALBERTc a Anheuser-Busch Eye Institute and Program in Cell and Molecular Biology, Saint Louis University, St. Louis, MO 63104, b Department of Molecular Biology, UMDNJSchool of Medicine, Stratford, NJ 08084, c Departments of Biochemistry and Ophthalmology, State University of New York (SUNY) at Buffalo, Buffalo, NY 14214, d Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa, FL 33612, U.S.A. References Bartlett, G. R. (1959). Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466–72. Bentz, J. and Ellens, H. (1988). Membrane fusion : kinetics and mechanisms. Colloids and Surf. 30, 65–112. Besharse, J. C. (1986). Photosensitive membrane turnover : differentiated membrane domains and cell-cell interaction. In : The retina : A model for cell biology studies. (Eds., Adler, R. and Farber, D.), Part I. Pp. 297–352, Academic Press : NY, U.S.A. Boesze-Battaglia, K. and Albert, A. D. (1989). Fatty acid composition of bovine rod outer segment plasma membrane. Exp. Eye Res. 49, 669–71. Boesze-Battaglia, K., Fliesler, S. J. and Albert, A. D. (1990). Relationship of cholesterol content to spatial distribution and age of disc membranes in retinal rod outer segments. J. Biol. Chem. 265, 18867–70. Boesze-Battaglia, K., Fliesler, S. J., Li, J., Young, J. E. and Yeagle, P. L. (1993). Retinal and retinol promote membrane fusion. Biochim. Biophys. Acta 1111, 256–62. * For correspondence at : Anheuser-Busch Eye Institute, 1755 South Grand Blvd., St. Louis, MO 63104-1540, U.S.A.
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Boesze-Battaglia, K., Hennessey, T. and Albert, A. D. (1989). Cholesterol heterogeneity in bovine retinal rod outer segment disk membranes. J. Biol. Chem. 264, 8151–5. Boesze-Battaglia, K. and Yeagle, P. L. (1992). Rod outer segment disc membranes are capable of fusion. Invest. Ophthalmol. Vis. Sci. 33, 484–93. Cullis, P. R. and Hope, M. R. (1985). Physical properties and functional roles of lipids in membranes. In : Biochemistry of lipids and membranes. (Eds Vance, D. E. and Vance, J. E.). Pp. 25–72, Benjamin}Cummings Publishing Co., Inc. : Menlo Park, U.S.A. Fliesler, S. J. (1979). Studies on the distribution and metabolism of sterols and other isoprenoids in bovine retina, Vols. 1 and 2. Ph.D. thesis, Rice University, Houston, TX, U.S.A. Fliesler, S. J. and Schroepfer, G. J., Jr. (1986). In vitro metabolism of mevalonic acid in the bovine retina. J. Neurochem. 46, 448–60. Fliesler, S. J., Florman, R. and Keller, R. K. (1995). Isoprenoid lipid metabolism in the retina : dynamics of squalene and cholesterol incorporation and turnover in frog rod outer segment membranes. Exp. Eye Res. 60, 57–69. Florman, R. W., Fliesler, S. J., Albert, A. D., Paw, Z., BoeszeBattaglia, K. and Keller, R. K. (1994). Isoprenoid lipid distribution in bovine ROS disc membranes and plasma membrane. Invest. Ophthalmol. Vis. Sci. (ARVO Suppl.) 35, 1461. Glaser, M. (1993). Lipid domains in biological membranes. Curr. Opin. Struct. Biol. 3, 475–81. Gruner, S. M. (1992). Nonlemellar lipid phases. In : The structure of biological membranes. (Ed. Yeagle, P. L.). Pp. 211–50, CRC Press : Boca Raton.
House, K., Badgett, D. and Albert, A. D. (1989). Cholesterol movement between bovine rod outer segment disk membranes and phospholipid vesicles. Exp. Eye Res. 49, 561–72. Keller, R. K. and Fliesler, S. J. (1990). Incorporation of squalene into rod outer segments. J. Biol. Chem. 265, 13709–12. Keller, R. K., Fliesler, S. J. and Nellis, S. (1988). Isoprenoid biosynthesis in the retina : quantitation of the sterol and dolichol biosynthetic pathways. J. Biol. Chem. 262, 2250–4. Litman, B. J. (1973). Lipid model membrane characterization of mixed phospholipid vesicles. Biochemistry 13, 2545–54. Lohner, K., Degovics, G., Laggner, P., Gnamusch, E. and Paltauf, F. (1993). Squalene promotes the formation of non-bilayer structures in phospholipid model membranes. Biochim. Biophys. Acta 1152, 69–77. Molday, R. S. and Molday, L. L. (1987). Differences in protein composition of bovine retinal rod outer segment disk and plasma membrane isolated by a ricin-golddextran density gradient perturbation method. J. Cell Biol. 105, 2589–601. Molday, R. S. and Molday, L. L. (1993). Isolation and characterization of rod outer segment disk and plasma membranes. In : Methods in neurosciences. (Ed. Hargrave, P. A.), Vol. 15. Pp. 131–50, Academic Press : Orlando, U.S.A. Schroepfer, G. J., Jr. (1981). Sterol biosynthesis. Ann. Rev. Biochem. 50, 585–621. Yeagle, P. L. (1993). Membrane fusion. In : The membranes of cells. (Ed. Yeagle, P. L.), 2nd Edition. Pp. 261–79, Academic Press : San Diego, U.S.A.
(Received Cleveland 4 June 1996 and accepted in revised form 2 July 1996)
LETTER TO THE EDITORS
Squalene is Localized to the Plasma Membrane in Bovine Retinal Rod Outer Segments Squalene, an isoprenoid hydrocarbon, is an obligatory, but transient, intermediate in the de novo synthesis of sterols in all eukaryotic cells (Schroepfer, 1981). In most cells and tissues (with the notable exception of liver), squalene represents an extremely minor percentage of the total lipids. We previously reported the unusual finding that the retina of the leopard frog (Rana pipiens), an animal commonly used in vision research, contains appreciable amounts of squalene, particularly in the rod outer segment (ROS) membranes (Keller, Fliesler and Nellis, 1988 ; Keller and Fliesler, 1990). The cholesterol}squalene mole ratio in frog ROS membranes is approximately 10 ; since cholesterol accounts for E 10 mol % of the total ROS membrane lipid, squalene represents E 1 mol %. Squalene is also present in bovine ROS membranes, but at levels approximately 100-fold lower than in the frog ROS (see below). Certain lipids have been shown to exhibit a preferential localization to the plasma membrane of the ROS, compared to the disc membranes. For example, cholesterol is enriched E 4-fold (normalized to total phospholipid content) in the plasma membrane relative to the majority of the mature disc membranes, and it also is enriched E 6-fold in the basal disc membranes relative to the older discs located toward the distal tip of the ROS (Boesze-Battaglia and Albert, 1989 ; Boesze-Battaglia, Hennessey and Albert, 1989 ; Boesze-Battaglia, Fliesler and Albert, 1990). Similarly, the ROS plasma membrane is relatively enriched (compared to the disc membranes) in the fatty acids C14 : 0 (E 14-fold), C18 : 2 (E 8-fold), and C18 : 3 (E 7-fold), whereas the mature discs are relatively enriched (compared to the plasma membrane) in C22 : 6 (E 7-fold) (Boesze-Battaglia and Albert, 1989). Since the relative distribution of squalene in these two distinct ROS membrane compartments is not known, the distribution of squalene, relative to cholesterol, was examined in highly purified bovine ROS plasma membranes and disc membranes. The results found suggest that most of the squalene in the bovine ROS is localized to the plasma membrane, while mature disc membranes are virtually devoid of squalene. Frozen, dark-adapted bovine retinas (50 retinas per preparation ; J. Lawson, Inc., Lincoln, NE, U.S.A.) were used for the preparation of purified ROS membranes (disc membranes and plasma membrane), using modifications of the method of Molday and Molday (1987), as described in detail previously (BoeszeBattaglia and Albert, 1989 ; Boesze-Battaglia, 0014-4835}97}02027904 $25.00}0}ey960201
Hennessey and Albert, 1989). Based upon both lipid phosphorus determination [using a modification of the method of Bartlett (1959), as described by Litman (1973)] and protein assay (micro-BCA method, per Pierce Chemical Co., Rockford, IL, U.S.A.), the recovered amount of ROS plasma membrane was 2–3 % that of the recovered disc membrane fraction. Aliquots of each membrane fraction (ca. 0±01 % of the disc membranes and ca. 10 % of the plasma membranes) were taken for SDS-PAGE analysis to confirm the characteristic protein pattern of each membrane fraction (Molday and Molday, 1987). Approximately 1 % of the disc membrane fraction and ca. 75 % of the plasma membrane fraction from each of three independent ROS membrane preparations were then subjected to saponification, extraction of the nonsaponifiable lipids with petroleum ether, and analysis of the nonsaponifiable lipids by reverse-phase HPLC, as described in detail elsewhere (Keller, Fliesler and Nellis, 1988 ; Keller and Fliesler, 1990). Serial dilutions of authentic cholesterol and squalene (Sigma Chemical Co., St. Louis, MO, U.S.A.) were used to calibrate the integrated detector response factors for each lipid (using detection at 210 nm), which permitted quantitation of the levels of those lipids in the biological samples analysed. Under the conditions employed, we could detect & 1 pmol of cholesterol (& 386 pg) and & 0±1 pmol of squalene (& 4±11 pg). The difference in detection sensitivity is due to the ten-fold greater extinction coefficient of squalene relative to that of cholesterol. Representative reverse-phase HPLC chromatograms of nonsaponifiable lipids from the disc membrane and plasma membrane fractions are shown in Fig. 1. The elution positions of cholesterol (designated ‘ C ’ ; retention time 12±0³0±1 min) and squalene (designated ‘ S ’ ; retention time 14±0³0±1 min) are indicated. Under the conditions employed, the cholesterol peak in the disc membrane fraction (panel A, 89 nmol) is nearly at full scale, whereas that corresponding to squalene is barely detectable by visual inspection (but is quantifiable by peak integration as 64 pmol). In this particular preparation, the cholesterol}squalene mole ratio was 1±4¬10$ ; the value (mean³..) obtained for all three preparations was 1±7³0±7¬10$. In striking contrast, the chromatogram obtained for the ROS plasma membranes (panel B ; using a sensitivity 16 times greater than in panel A) exhibits a peak corresponding to squalene (10 pmol) that is greater in height than the peak corresponding to cholesterol (55 # 1997 Academic Press Limited
280
LETTER TO THE EDITORS (A)
(B)
6.72
c
0
5
13.96
9.80
12.00
s
s
14.06
Relative absorbance (210 nm)
11.95
c
10 15 0 5 Retention time (min)
10
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F. 1. Reverse-phase HPLC chromatograms of nonsaponifiable lipids isolated from (A) ROS disc membranes and (B) ROS plasma membrane. The elution positions of cholesterol (C) and squalene (S) are indicated. Note that the detector sensitivity employed for obtaining the data in panel (B) was 16-fold greater than that employed to obtain the data shown in panel (A) (relative response, absorbance at 210 nm).
pmol). The mole ratio of cholesterol}squalene in this plasma membrane preparation was 5±5 ; for the three preparations analysed, this value was 6±3³2±9. Thus, when one normalizes the squalene content relative to that of cholesterol (mol : mol), the ROS plasma membrane fraction exhibits nearly a 300-fold enrichment in squalene compared to the bulk of the mature disc membranes. Using these empirically determined ratios and the values previously determined for the cholesterol} phospholipid mole ratio of the bovine ROS plasma membrane (0±38) and the average disc membrane population (0±11) (Boesze-Battaglia, Hennessey and Albert, 1989), the calculated average values for the squalene}phospholipid mole ratio of the ROS plasma membrane and disc membranes are 6±1¬10−# and 6±5¬10−&, respectively. This would suggest almost a 10$-fold concentration difference in squalene between the plasma membrane and mature disc membranes. Since the disc membranes account for about 15 times more of the total ROS membrane mass than does the plasma membrane, the preferential localization of squalene to the plasma membrane, relative to the discs, is on the order of 70-fold. Comparing this value with the compositional differences reported for ROS plasma membrane and disc membranes with respect to cholesterol and various phospholipid acyl chains, the squalene concentration difference is the largest observed for any lipid species in ROS membranes (i.e., on the order of 10-fold greater than other lipid differences).
Although bovine ROS are clearly squalene-deficient compared with frog ROS, we employed bovine ROS in this study because the methods available for separating and purifying the plasma membrane and disc membrane fractions to near homogeneity have been established for bovine, but not frog, retina. Due to the relatively large tissue mass, one can obtain sufficient yields of ROS plasma membrane from bovine retinas to perform detailed biochemical analyses. In contrast, our repeated attempts to obtain a similar degree of fractionation and purification of ROS plasma membranes from frog retina (typically using 200–250 retinas per preparation), and in sufficient yields for similar biochemical analyses, have been unsuccessful. Hence, although considerably more is known about isoprenoid lipid metabolism in the frog retina than in any other species, and although the frog ROS exhibits a profound enrichment in squalene, we have not been able to determine the relative distribution of squalene in the membranes of the frog ROS. The squalene found in ROS membranes most likely arises from de novo synthesis in the rod cells. The formation of squalene in the retina from common, radiolabeled de novo precursors (e.g., [$H]acetate, [$H]mevalonate) has been demonstrated both in vitro and in vivo in frog and cow eyes (Fliesler, 1979 ; Fliesler and Schroepfer, 1986 ; Keller, Fliesler and Nellis, 1988 ; Keller and Fliesler, 1990 ; Fliesler, Florman and Keller, 1995). In all those studies, radiolabeled squalene, rather than cholesterol, was by far the predominant nonsaponifiable lipid product formed. In the frog retina, a substantial portion of the total radiolabeled squalene formed is subsequently transported to and incorporated into the ROS membranes, where it turns over independent of and without conversion to cholesterol (Keller and Fliesler, 1990 ; Fliesler, Florman and Keller, 1995). The incorporation of newly synthesized squalene into the ROS has been observed also in the bovine retina in vitro (Florman et al., 1994). As discussed in detail previously (see Fliesler, Florman, and Keller, 1995), the retinal pigment epithelium is not likely to contribute significantly to the squalene mass in the ROS. The presence of appreciable steady-state amounts of squalene, and its preferential segregation in the plasma membrane vs. the disc membranes of the ROS, begs at least two questions : (1) What is the biological role of squalene in the ROS ? and (2) What are the driving forces that promote the relative segregation of squalene into the plasma membrane ? In regard to the first question, prior studies on squalene metabolism in the frog retina would suggest that it is not serving its classic biogenic role as a cholesterol precursor, since squalene is not converted to cholesterol in the ROS (Fliesler, Florman and Keller, 1995). Clues to the potential role of squalene in the ROS may be derived from studies concerning its biophysical effects in artificial membranes. In this regard, Lohner and coworkers (Lohner
LETTER TO THE EDITORS
et al., 1993) have shown that squalene, at levels & 6 mol % of the total lipid, can destabilize the bilayer (‘ lamellar ’ phase) structure of artificial phospholipid membranes, thereby promoting the formation of nonbilayer structures, e.g., the ‘ inverse hexagonal ’ (HII) phase. Such alternate phase structures have been shown to have profound effects on the properties and functions of biological membranes, including the translational and rotational mobility of membrane proteins, the activity of membrane enzymes, and ion permeability (reviewed in Cullis and Hope, 1985 ; Gruner, 1992 ; Glaser, 1993). Our calculations indicate that the average squalene content of the bovine ROS plasma membrane approaches 6 mol % of the total lipid. While this concentration of squalene may be barely sufficient to perturb membrane structure (assuming isotropic distribution), nonhomogeneous segregation of squalene could provide localized concentrations of squalene sufficient to promote non-bilayer phases in discrete membrane domains, without altering the bulk, longrange bilayer structure. One potential consequence of such localized alternate membrane phase structure is the promotion of membrane fusion events (reviewed in Bentz and Ellens, 1988 ; Yeagle, 1993), which are apparently requisite in the processes of disc morphogenesis and disc shedding (reviewed by Besharse, 1986). Recently, it has been shown that the ability of ROS disc membranes to undergo fusion is correlated with the presence of non-bilayer, ‘ isotropic ’ phases, which can be promoted by increasing the molar concentration of retinol or retinal in the membrane (Boesze-Battaglia and Yeagle, 1992 ; Boesze-Battaglia et al., 1993). Hence, the distribution and concentration of free isoprenoids and}or retinoids might be important in controlling the spatial and temporal frequency of membrane fusion events involved in ROS membrane renewal and turnover. With regard to the second question, the answer is not readily apparent. Although squalene tends to partition preferentially into ‘ liquid-crystalline ’, as opposed to ‘ gel ’, phases in aqueous phospholipid dispersions (Lohner et al., 1993), both the disc membranes and plasma membrane are likely to exist in the liquid-crystalline state at physiological temperatures (see Cullis and Hope, 1985). In contrast to cholesterol, which can exchange between physically discontinuous membranes across an aqueous space (House, Badgett and Albert, 1989) and apparently does so as the disc membranes are displaced progressively toward the distal tip of the ROS (BoeszeBattaglia, Hennessey and Albert, 1989 ; BoeszeBattaglia, Fliesler, and Albert, 1990), squalene (a hydrocarbon) would not be expected to partition between membranes via an aqueous environment. Hence, the ‘ molecular segregation ’ of squalene must occur during or immediately prior to the maturation of new discs near the base of the ROS. A similar process is likely involved in the absolute sorting of various
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membrane proteins to either the plasma membrane (e.g., the GLUT-1 glucose transporter, the cGMP-gated channel and channel-associated protein, the Na+}K+}Ca#+ exchanger) or the disc membranes (e.g., ROM-1, peripherin}rds, ‘ rim protein ’) (see Molday and Molday, 1987, 1993). However, the details of such sorting mechanisms remain to be elucidated. Acknowledgements This study was supported by U.S.P.H.S. (NEI}NIH) grants EY07361 (SJF and RKK), EY10420 (KB-B), and EY03328 (ADA), and by an unrestricted departmental grant from Research to Prevent Blindness, Inc. (SJF).
STEVEN J. FLIESLERa* KATHLEEN BOESZE-BATTAGLIAb ZOPHIA PAWc R. KENNEDY KELLERd ARLENE D. ALBERTc a Anheuser-Busch Eye Institute and Program in Cell and Molecular Biology, Saint Louis University, St. Louis, MO 63104, b Department of Molecular Biology, UMDNJSchool of Medicine, Stratford, NJ 08084, c Departments of Biochemistry and Ophthalmology, State University of New York (SUNY) at Buffalo, Buffalo, NY 14214, d Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa, FL 33612, U.S.A. References Bartlett, G. R. (1959). Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466–72. Bentz, J. and Ellens, H. (1988). Membrane fusion : kinetics and mechanisms. Colloids and Surf. 30, 65–112. Besharse, J. C. (1986). Photosensitive membrane turnover : differentiated membrane domains and cell-cell interaction. In : The retina : A model for cell biology studies. (Eds., Adler, R. and Farber, D.), Part I. Pp. 297–352, Academic Press : NY, U.S.A. Boesze-Battaglia, K. and Albert, A. D. (1989). Fatty acid composition of bovine rod outer segment plasma membrane. Exp. Eye Res. 49, 669–71. Boesze-Battaglia, K., Fliesler, S. J. and Albert, A. D. (1990). Relationship of cholesterol content to spatial distribution and age of disc membranes in retinal rod outer segments. J. Biol. Chem. 265, 18867–70. Boesze-Battaglia, K., Fliesler, S. J., Li, J., Young, J. E. and Yeagle, P. L. (1993). Retinal and retinol promote membrane fusion. Biochim. Biophys. Acta 1111, 256–62. * For correspondence at : Anheuser-Busch Eye Institute, 1755 South Grand Blvd., St. Louis, MO 63104-1540, U.S.A.
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(Received Cleveland 4 June 1996 and accepted in revised form 2 July 1996)