Biochimie (1991) 73, 1303-1310 © Soci6t6 frangaise de biochimie et biologie mol6culaire / Elsevier, Paris
1303
Modulation of membrane function by cholesterol PL
Yeagle
Department of Biochemistry, 140 Farber Hall, University at Buffalo (SUNY) School of Medicine, Buffalo, NY 14214 USA (Received 24 May 1991; accepted 17 September 1991)
Summary - - The molecular basis for the essential role of cholesterol in mammalian (and other cholesterol-requiring) cells has long been the object of intense interest. Cholesterol has been found to modulate the function of membrane proteins critical to cellular function. Current literature supports two mechanisms for this modulation. In one mechanism, the requirement of "free volume' by integral membrane proteins for conformational changes as part of their functional cycle is antagonized by the presence of high levels of cholesterol in the membrane. In the other mechanism, the sterol modulates membrane protein function through direct sterol-protein interactions. This mechanism provides an explanation for the stimulation of the activity of important membrane proteins and for the essential requirement of a structurally-specific sterol for cell viability. In some cases, these latter membrane proteins exhibit little or no activity in the absence of the specific sterol required for growth of that cell type. The specific sterol required varies from one cell type to another and is unrelated to the ability of that sterol to affect the bulk properties of the membrane. cholesterol / cholesterol-protein interactions / membrane / cholesterol regulation of enzymes Cholesterol has long been known to be an essential component of m a m m a l i a n cells. Yet despite much study, the role cholesterol plays in m a m m a l i a n cell biology has remained a mystery. Without cholesterol, m a m m a l i a n cells cannot experience normal growth. As a consequence, most m a m m a l i a n cells are capable of making their own cholesterol. M a n y steps are involved in cholesterol biosynthesis. Even after the synthesis of lanosterol, 18 enzymatically catalyzed steps remain to produce cholesterol [1]. Much valuable cellular energy is therefore utilized in the complex biosynthetic pathway to produce the particular chemical structure of cholesterol. W h y this occurs is not fully understood. Bloch has suggested that evolutionary pressure for a more biologically competent sterol led to the development of the pathway from lanosterol to cholesterol [2]. However, the molecular details describing why the cholesterol structure is required for biological competence (for example, in m a m m a l i a n cells) have not yet been fully described.
Structural r e q u i r e m e n t s for sterols in cell biology Some studies are available on specificity for sterol structure by sterol-requiring cells. In vitro experi-
ments have shown that lanosterol cannot fully substitute for cholesterol as the essential sterol for mycoplasma cell function [3]. In particular, Mycoplasma mycoides can be adapted to grow on low cholesterol media. However, they cannot grow in the total absence of cholesterol in the medium, since they do not make their own cholesterol and cholesterol is required for cell growth and function. Supplementation in the medium of lanosterol will not support cell growth in the absence of cholesterol. However, cell growth will occur at nearly the same rate in cells fed low cholesterol levels supplemented with high (relatively) lanosterol levels, as in cells fed high (relatively) cholesterol levels [4]. Thus, cholesterol appears both adequate and necessary for minimal cellular function in these mycoplasma, while for optim u m cellular function higher membrane stero! content is required but w~thout the structural specificity associated with the requirement for minimal cellular function. This sterol synergism has pointed to a special role for cholesterol in supporting cell growth in which the particular chemical structure of cholesterol is required for mycoplasma. The requirement of cholesterol for normal function of m y c o p l a s m a is mirrored in yeast by an analogous requirement for ergosterol [3]. Yet one steroi cannot substitute for the other; cholesterol cannot fully
! 304
PL Yeagle
substitute for ergosterol in yeast, and ergosterol cannot support normal mammalian cellular function. Anaerobic growth of S a c c h a r o m y c e s cerevisiae requires ergosterol supplement to the culture medium. Supplementation only with cholesterol will not support normal growth [5]. Yet cholesterol is more effective at modifying the properties of lipid bilayers than is ergosterol (see below). These data point to a specific sterol recognition interaction, crucial to normal cellular function, that is different in yeast than it is in mycoplasma. The requirement for cholesterol in mammalian cells is not well understood due to the paucity of available mammalian sterol auxotrophes. Therefore, other methods have been used to explore the role of cholesterol. In mammalian cells, inhibition of cellular cholesterol biosynthesis inhibits cell growth [6]. In addition to the role of metabolic products from mevalonate other than cholesterol in cell growth, cholesterol itself played a role since the addition of cholesterol in the culture medium would restore cell growth [7]. Data such as these suggest that a unique sterol structure leads to biological competence in each sterol-requiring organism. What defines that biological competence is central to the discussion in this article. From this analysis emerges an hypothesis for the essential role of cholesterol in mammalian (and other cholesterol-requiring) cells.
Physical effects of cholesterol on membrane structure Cholesterol has been the subject of extensive investigation for many years [8]. Cholesterol is predominantly found in the membranes of cells, due to its largely hydrophobic structure [9]. Much effort has been expended exploring the fascinating effects cholesterol has on the bulk properties of lipid bilayers, including cholesterol effects on permeability [ 10] and molecular ordering [11, 12], lateral phase separations [13], reduction of the enthalpy of phospholipid phase transitions from the gel to the liquid crystalline state [14], to name just a few. As will be seen in the following, this line of study has provided information about some of the effects of cholesterol on biological membrane function (for a review, see [15]). However, the structural (with respect to sterol) specificity of these physical effects does not mirror the considerable structural specificity of some of the biological effects for sterols. And as noted above, some patterns observed in the physical data have no correlation at all with the biological requirement for sterol structures ( i e , t h e yeast data referred to above). Therefore, the search for the role of cholesterol in sterol-requiring cells has broadened to encompass more specific struc-
tural effects that may arise from the interaction between sterols and membrane proteins.
Modulation of biological function of membranes by cholesterol Since the primary location of cholesterol in cells is in the membranes of the cells, the ability of cholesterol to modify membrane function has been the focus of much interest. In particular, the modulation by cholesterol of the function of membrane proteins has been examined, both by modifying the cholesterol content of the nativc membranes in which the protein is found, and by reconstituting the membrane proteins into membranes of defined lipid content. Studies of this sort have led to three different classes of observations: 1) An increase in the level of cholesterol in the membrane leads to a proportionate decrease in membrane protein function. An example can be seen in figure 1. In this example, the equilibrium constant for the Meta I-Meta II transition of rhodopsin was measured as a function of the cholesterol content of reconstituted membranes [16]. The data show an inverse relationship between the function of the membrane protein and the cholesterol content of the membrane. For rhodopsin, cholesterol apparently acts as a negative modulator, or an inhibitor, at high cholesterol levels. Similar inhibitory effects of cholesterol have been observed on rhodopsin activation of the cyclic GMP cascade [17], alkaline phosphatase [18], UDP-glucuronosyltransferase [19], thymidine transport [20], and anion transport [21 ]. 2) An increase in the level of cholesterol in the membrane leads to a proportionate increase in membrane protein function. This ,,,,,"'m r,u,. . . . .~,.,.,, ... ,,~ ,~,.~. . . membrane cholesterol levels for the Na+-K+-ATPase in figure 2. From a cholesterol/phospholipid mol ratio of 0 to about 0.35, an increase in cholesterol in the membrane leads to an increase in the ATP hydrolyzing activity of this enzyme in the modified native membrane. Stimulation of other membrane functions by cholesterol has been observed, including Na+-Ca 2÷ exchange [22], ATP-ADP exchange [23], carder mediated lactate transport [24] and the acetyl choline receptor [25, 26]. (At high membrane cholesterol levels, inhibition is observed as as described in the examples in 1.) 3) Some membrane functions appear to be insensitive to cholesterol levels in the membrane. Figure 3 shows data for the ATPase activity of the rabbit sarcoplasmic reticulum. As membrane cholesterol content was varied over a wide range, no alteration in ATPase activity was observed. Other membrane functions such as sucrase, lactase and maltase activities of the rat intestinal microvillus are apparently unaffected by alterations in the membrane cholesterol level [18].
Essential role of cholesterol in cells 0.8
0.6
!
1305
120 lOO
8o E =
0.4
60
E ¢~
40
E
0.2
20
0.0
I
0
"
I
'
I
10 20 30 Mole % Cholesterol
•
Fig 1. Effect of membrane cholesterol on the equilibrium constant for the Meta I-Meta II transition of bovine rhodopsin in reconstituted systems at 20°C as a function of cholesterol content of the membranes (mol % with respect to the total lipid content of the membranes). Data replotted from [ 16].
The challenge is to find an explanation for these varied observations. The approach to be taken here is to identify the known effects of cholesterol on membrane structure and to use these properties to explain the observed effects of cholesterol on m e m brane function. A review of the available literature indicates that there are at least two general classes of interactions in which cholesterol can engage while in a membrane.
I
•
10
40
I
•
20
I
30
"
I
"
40
I
50
"
60
M o l e % cholesterol
Fig 2. Effect of membrane cholesterol on the ATPase activity of the bovine kidney Na+-K+-ATPase. Cholesterol content was modified by incubation of the native membranes with lipid vesicles, with or without cholesterol, which resulted in intermembrane cholesterol transfer and alterations in the cholesterol level in the native membrane. Data replotted from [42].
[,.,
1303
Modulation of membrane function by cholesterol PL
Yeagle
Department of Biochemistry, 140 Farber Hall, University at Buffalo (SUNY) School of Medicine, Buffalo, NY 14214 USA (Received 24 May 1991; accepted 17 September 1991)
Summary - - The molecular basis for the essential role of cholesterol in mammalian (and other cholesterol-requiring) cells has long been the object of intense interest. Cholesterol has been found to modulate the function of membrane proteins critical to cellular function. Current literature supports two mechanisms for this modulation. In one mechanism, the requirement of "free volume' by integral membrane proteins for conformational changes as part of their functional cycle is antagonized by the presence of high levels of cholesterol in the membrane. In the other mechanism, the sterol modulates membrane protein function through direct sterol-protein interactions. This mechanism provides an explanation for the stimulation of the activity of important membrane proteins and for the essential requirement of a structurally-specific sterol for cell viability. In some cases, these latter membrane proteins exhibit little or no activity in the absence of the specific sterol required for growth of that cell type. The specific sterol required varies from one cell type to another and is unrelated to the ability of that sterol to affect the bulk properties of the membrane. cholesterol / cholesterol-protein interactions / membrane / cholesterol regulation of enzymes Cholesterol has long been known to be an essential component of m a m m a l i a n cells. Yet despite much study, the role cholesterol plays in m a m m a l i a n cell biology has remained a mystery. Without cholesterol, m a m m a l i a n cells cannot experience normal growth. As a consequence, most m a m m a l i a n cells are capable of making their own cholesterol. M a n y steps are involved in cholesterol biosynthesis. Even after the synthesis of lanosterol, 18 enzymatically catalyzed steps remain to produce cholesterol [1]. Much valuable cellular energy is therefore utilized in the complex biosynthetic pathway to produce the particular chemical structure of cholesterol. W h y this occurs is not fully understood. Bloch has suggested that evolutionary pressure for a more biologically competent sterol led to the development of the pathway from lanosterol to cholesterol [2]. However, the molecular details describing why the cholesterol structure is required for biological competence (for example, in m a m m a l i a n cells) have not yet been fully described.
Structural r e q u i r e m e n t s for sterols in cell biology Some studies are available on specificity for sterol structure by sterol-requiring cells. In vitro experi-
ments have shown that lanosterol cannot fully substitute for cholesterol as the essential sterol for mycoplasma cell function [3]. In particular, Mycoplasma mycoides can be adapted to grow on low cholesterol media. However, they cannot grow in the total absence of cholesterol in the medium, since they do not make their own cholesterol and cholesterol is required for cell growth and function. Supplementation in the medium of lanosterol will not support cell growth in the absence of cholesterol. However, cell growth will occur at nearly the same rate in cells fed low cholesterol levels supplemented with high (relatively) lanosterol levels, as in cells fed high (relatively) cholesterol levels [4]. Thus, cholesterol appears both adequate and necessary for minimal cellular function in these mycoplasma, while for optim u m cellular function higher membrane stero! content is required but w~thout the structural specificity associated with the requirement for minimal cellular function. This sterol synergism has pointed to a special role for cholesterol in supporting cell growth in which the particular chemical structure of cholesterol is required for mycoplasma. The requirement of cholesterol for normal function of m y c o p l a s m a is mirrored in yeast by an analogous requirement for ergosterol [3]. Yet one steroi cannot substitute for the other; cholesterol cannot fully
! 304
PL Yeagle
substitute for ergosterol in yeast, and ergosterol cannot support normal mammalian cellular function. Anaerobic growth of S a c c h a r o m y c e s cerevisiae requires ergosterol supplement to the culture medium. Supplementation only with cholesterol will not support normal growth [5]. Yet cholesterol is more effective at modifying the properties of lipid bilayers than is ergosterol (see below). These data point to a specific sterol recognition interaction, crucial to normal cellular function, that is different in yeast than it is in mycoplasma. The requirement for cholesterol in mammalian cells is not well understood due to the paucity of available mammalian sterol auxotrophes. Therefore, other methods have been used to explore the role of cholesterol. In mammalian cells, inhibition of cellular cholesterol biosynthesis inhibits cell growth [6]. In addition to the role of metabolic products from mevalonate other than cholesterol in cell growth, cholesterol itself played a role since the addition of cholesterol in the culture medium would restore cell growth [7]. Data such as these suggest that a unique sterol structure leads to biological competence in each sterol-requiring organism. What defines that biological competence is central to the discussion in this article. From this analysis emerges an hypothesis for the essential role of cholesterol in mammalian (and other cholesterol-requiring) cells.
Physical effects of cholesterol on membrane structure Cholesterol has been the subject of extensive investigation for many years [8]. Cholesterol is predominantly found in the membranes of cells, due to its largely hydrophobic structure [9]. Much effort has been expended exploring the fascinating effects cholesterol has on the bulk properties of lipid bilayers, including cholesterol effects on permeability [ 10] and molecular ordering [11, 12], lateral phase separations [13], reduction of the enthalpy of phospholipid phase transitions from the gel to the liquid crystalline state [14], to name just a few. As will be seen in the following, this line of study has provided information about some of the effects of cholesterol on biological membrane function (for a review, see [15]). However, the structural (with respect to sterol) specificity of these physical effects does not mirror the considerable structural specificity of some of the biological effects for sterols. And as noted above, some patterns observed in the physical data have no correlation at all with the biological requirement for sterol structures ( i e , t h e yeast data referred to above). Therefore, the search for the role of cholesterol in sterol-requiring cells has broadened to encompass more specific struc-
tural effects that may arise from the interaction between sterols and membrane proteins.
Modulation of biological function of membranes by cholesterol Since the primary location of cholesterol in cells is in the membranes of the cells, the ability of cholesterol to modify membrane function has been the focus of much interest. In particular, the modulation by cholesterol of the function of membrane proteins has been examined, both by modifying the cholesterol content of the nativc membranes in which the protein is found, and by reconstituting the membrane proteins into membranes of defined lipid content. Studies of this sort have led to three different classes of observations: 1) An increase in the level of cholesterol in the membrane leads to a proportionate decrease in membrane protein function. An example can be seen in figure 1. In this example, the equilibrium constant for the Meta I-Meta II transition of rhodopsin was measured as a function of the cholesterol content of reconstituted membranes [16]. The data show an inverse relationship between the function of the membrane protein and the cholesterol content of the membrane. For rhodopsin, cholesterol apparently acts as a negative modulator, or an inhibitor, at high cholesterol levels. Similar inhibitory effects of cholesterol have been observed on rhodopsin activation of the cyclic GMP cascade [17], alkaline phosphatase [18], UDP-glucuronosyltransferase [19], thymidine transport [20], and anion transport [21 ]. 2) An increase in the level of cholesterol in the membrane leads to a proportionate increase in membrane protein function. This ,,,,,"'m r,u,. . . . .~,.,.,, ... ,,~ ,~,.~. . . membrane cholesterol levels for the Na+-K+-ATPase in figure 2. From a cholesterol/phospholipid mol ratio of 0 to about 0.35, an increase in cholesterol in the membrane leads to an increase in the ATP hydrolyzing activity of this enzyme in the modified native membrane. Stimulation of other membrane functions by cholesterol has been observed, including Na+-Ca 2÷ exchange [22], ATP-ADP exchange [23], carder mediated lactate transport [24] and the acetyl choline receptor [25, 26]. (At high membrane cholesterol levels, inhibition is observed as as described in the examples in 1.) 3) Some membrane functions appear to be insensitive to cholesterol levels in the membrane. Figure 3 shows data for the ATPase activity of the rabbit sarcoplasmic reticulum. As membrane cholesterol content was varied over a wide range, no alteration in ATPase activity was observed. Other membrane functions such as sucrase, lactase and maltase activities of the rat intestinal microvillus are apparently unaffected by alterations in the membrane cholesterol level [18].
Essential role of cholesterol in cells 0.8
0.6
!
1305
120 lOO
8o E =
0.4
60
E ¢~
40
E
0.2
20
0.0
I
0
"
I
'
I
10 20 30 Mole % Cholesterol
•
Fig 1. Effect of membrane cholesterol on the equilibrium constant for the Meta I-Meta II transition of bovine rhodopsin in reconstituted systems at 20°C as a function of cholesterol content of the membranes (mol % with respect to the total lipid content of the membranes). Data replotted from [ 16].
The challenge is to find an explanation for these varied observations. The approach to be taken here is to identify the known effects of cholesterol on membrane structure and to use these properties to explain the observed effects of cholesterol on m e m brane function. A review of the available literature indicates that there are at least two general classes of interactions in which cholesterol can engage while in a membrane.
I
•
10
40
I
•
20
I
30
"
I
"
40
I
50
"
60
M o l e % cholesterol
Fig 2. Effect of membrane cholesterol on the ATPase activity of the bovine kidney Na+-K+-ATPase. Cholesterol content was modified by incubation of the native membranes with lipid vesicles, with or without cholesterol, which resulted in intermembrane cholesterol transfer and alterations in the cholesterol level in the native membrane. Data replotted from [42].
[,.,
