The structure of membrane transport systems

TIB8V- January I9 76

I1

The structure of membrane transport systems Guido Guidotti Solutes are carried across eukaryotic plasma membranes by oligomeric glycoproteins which span the cell membranes and conduct transport by undergoing conformational changes.

The membranes of cells are composed of proteins and lipids [l]. The lipid bilayer is impermeable to almost all solutes (except for gases). The proteins carry out transport of most solutes across the membranes. The central question is: how do proteins execute this function? To a first approximation the answer is that in plasma membranes of eukaryotic cells transport is catalyzed by oligomeric glycoproteins which span the bilayer and transport solutes by undergoing conformational changes. This statement is deduced from information available on five membrane proteins which either are involved in, or are thought to be involved in, solute transport (Table I). (Na+ +K+)-ATPase

This enzyme is involved in the transport of Na+ and K+ across the plasma membrane of all eukaryotic cells. The purified enzyme is composed of two polypeptide chains - one with a mass of 90,000 daltons (a chain) and the other, a glycoprotein, with a mass of 40,000 daltons (/3 chain) [2]. The r and fi chains are probably present in a 1 to 1 molar ratio, although the evidence on this point is ambiguous [2]. TABLE I --

____

___

Cross-linking studies are consistent with the view that there is close contact between an ‘Aand b chain (a/l subunit). An a/3 subunit is also obtained when the purified enzyme is solubilized in Triton X-100 [3]. Whether or not the a/l unit is functionally active is not known. More recent crosslinking studies [4] suggest that two /? chains are in close contact. Therefore the structure of the functional enzyme can be deduced to be a&, with a protein molecular weight of 260,000. There are one to two moles of phosphate bound to 260,OOOg of protein (hence one to two phosphorylation sites per a$2 complex) [2] and the number of binding sites for cardiac glycosides per a& complex varies between 1 and 2 [2]. This variability may be due simply to experimental error; on the other hand further evidence that there is one cardiac glycoside binding site per two phosphorylation sites would support the proposal that the a& structure is the unit of function. It has been reported that antibodies to the glycoprotein decrease the enzymatic activity; this report has raised the possibility that this chain may be involved directly in the function, but there is no conclusive

._ Anionexchange protein

Acetylcholine receptor

a-100,000

a-90,000

a- 40,000 B- 48,000 y- 58,000 6- 64,000 E-105.000

a-38.000

a

4B --El?

a

(Na++K+)-Cat+ATPase ATPase Molecular weights of component polypeptides

8-40.000

Glycoproteins

B

a

Probable structure and molecular weight of the protein part of the enzyme

a282

(a2?)

260,000

(2oo,ooo?)

Transmembrane

a

arrangement

a-90.000

~o,ooo

Rhodopsin

to ~4?)

62 or a2B2

(a2

240,ooo

(76,OtW 152,000?)

a

a

Detergent binding (mg/mg of protein)

0.28

0.20

0.77

0.7

1.10

Relative hydrophobic surface area of subunit

0.20-0.24

0.2-0.25

0.5-0.65

0.54.6

0.54

G.G. is 01 The Biologicid ktborotories, Cambridge, Massachusetts 02138. USA

evidence in this regard [5]. The large polypeptide is clearly required for the function : it is phosphorylated on the cytoplasmic surface of the membrane by ATP. it has the binding site for cardiac glycosides on the outer surface of the membrane, and it spans the bilayer [4]. The trans-membrane arrangement of the protein is supported by the observation that the protein binds weak detergents tightly and that consequently a considerable fraction of its surface is hydrophobic [3]. This is the part of the enzyme that is in contact with the non-polar part of the lipid bilayer. Ca* +-ATPase

This enzyme is located in the membranes of sarcoplasmic reticulum and it catalyzes the ATP-dependent Ca* +-transport from the cytoplasm into the space defined by the sarcoplasmic reticulum [6]. The purified enzyme is composed mainly of a polypeptide with an apparent mass of 102,000 daltons [7,8], but there is a substantial although variable amount of a higher aggregate in the 200,000 dalton range (see Fig. 6 of ref. [7] and Fig. 1, inset, fraction 9 of ref. [8]. While analyses for neutral sugars suggest that the 102,000 dalt& polypeptide may be a glycoprotein (L. Waxman, personal communication), further information is required on this point. The presence of components with a mass of 200,000 daltons in the purified membranes and the increase in the 200,000 molecular weight polypeptide brought about by cross-linking reagents (which also generate material with masses of 300,000 and 400,000 daltons) (G. Giotta, personal communication) suggest that the functional unit of the enzyme is an a2 structure. There is no direct demonstration that the polypeptide chain spans the bilayer, but it does bind detergent in the same way that the (Nat + K+)-dependent ATPase does [3]. There are other striking similarities between these two enzymes [9]. Anion-exchange protein

Anion exchange across the membrane of the red blood cell is a rapid process with the characteristics of facilitated exchange diffusion [ 10). The transport system has not been isolated in pure form. However, inactivation of transport is stoichiometric with the covalent attachment of a derivative of sulfanilate [l l] and of stilbene disulfonate [12] to a polypeptide with a mass of 100,000 daltons (band 3). The polypeptide or the group of polypeptides with this molecular weight are glycoproteins with a carbohydrate composition considerably different from that of the major red cell glycoprotein (PAS I) [l 11; they also have the receptor for Concanavalin A. The molecular weight of the protein

obtained by solubilization of the membrane with Triton X-100 is 180,000 [3], suggesting that the structure of the native protein is at least a dimer (x2). This conclusion is supported by cross-linking studies [13]. While it is possible that the material in the 100,000 molecular weight region is heterogeneous, it is clear that all these polypeptides span the bilayer as first demonstrated by Bretscher. Indeed, the transmembrane arrangement of this protein is supported by the finding that a large amount of weak detergent is bound by the protein, suggesting that a large fraction of its surface area is hydrophobic. Acetylcholine

receptor

The acetylcholine receptor changes the permeability of the membrane to ions when it forms a complex with acetylcholine [15]. The receptor has been isolated from the electric organ of several species of electric fish. The purified material has an apparent molecular weight of 250,000 for the protein part [16]; this amount of protein binds 2 moles of toxin and one mole of cholinergic ligand [ 151.The correct polypeptide composition of the purified receptor is not obvious. All preparations seem to contain a 40,000 molecular weight polypeptide which can be afIinity labelled [ 171. There are, however, varying amounts of other polypeptides with approximate masses of 48,000-50,000, 53,OOC-58,000, and 64,000 daltons, and a small amount of a 105,000 dalton component depending on the tissue and the experimentor [15]. According to Rafter-y [ 181, the receptor is a glycoprotein. The 40,000 dalton component is certainly a part of the receptor [17]; the other polypeptides may either be part of contaminating proteins, part of the actual receptor, or the variability in number and amount of polypeptides may be a result of proteolysis of a single 100,000-l 50,000 dalton polypeptide. In any event the mass per toxin binding site is in the range lOO,OoO-150,000 daltons. The basic structure of the receptor probably is a dimer of 120,000 molecular weight polypeptides. Giotta has suggested that the smaller polypeptides are probably derived from the larger ones by proteolysis, either during folding and positioning of the protein in the bilayer, or during the process of isolation (G. Giotta, personal communication). There is no evidence to indicate whether or not the protein spans the bilayer. However, the receptor does bind a considerable amount of detergent [ 161, and thus a transmembrane arrangement is very likely. Rhodopsin Rhodopsin is the visual pigment which upon interaction with light causes a change

in the permeability of the plasma membrane of the cell in which it resides [19]. In vertebrate rods, rhodopsin is found in the membrane of intracellular vesicles called discs. It is composed of a glycoprotein with a mass of 36,000-38,000 daltons, and of a pigment retinal [20]. The structure of the native molecule in the disc membrane is not known, but the results of molecular weight determinations in mild detergent [3] suggest that the structure is at least an ~(2dimer. This number is also consistent with the observation that the number of intramembrane particles, which must correspond to rhodopsin molecules, is less by a factor of 2-4 than the number of rhodopsin monomers per membrane [21]. Considerable experimental evidence, reviewed by Cone [22], suggests that the rhodopsin molecule spans the bilayer. While there is no direct evidence for this conclusion, rhodopsin does bind a large amount ofdetergent [3] and thus does have a large hydrophobic surface which supports the conclusion that the molecule has a transmembrane orientation. General remaiks So far, only five membrane transport systems have been isolated from eukaryotic systems and well characterized. It is striking that all five of them appear to have the same characteristics: they are transmembrane oligomeric glycoproteins. Here, I suggest reasons for these general characteristics of membrane transport systems. (1) The proteins span the bilayer (are transmembrane proteins) because they catalyze transport by undergoing small conformational changes rather than by diffusing backwards and forward across the bilayer. It might be argued that a trans-

membrane protein could transport solutes by rotating through the bilayer around an axis parallel to the membrane. However, this possibility is unlikely because of the asymmetric arrangement of the transport systems ((Na * + K +)-ATPase, anionexchange system, rhodopsin) with regard to the inner and outer surface of the membrane [4,14,23]. Therefore, the transport proteins are embedded in a fixed and timeindependent asymmetric orientation in the bilayer. Accordingly, they catalyze transport through small conformational changes. (2) The proteins are oligomeric either because the active site for transport is produced at the interfaces between subunits (much like the binding site for organic phosphates is, at the adjacent surfaces between the /I chains of hemoglobin) or because allosteric control is more easily realized in oligomeric proteins, or for both reasons. Since the proteins are arranged asymmetrically with regard to the inner and outer surface of the membrane (see point 1 above), the axis of symmetry of the oligomeric protein must be perpendicular to the surface of the membrane. For dimeric proteins, this condition can be achieved by homologous or heterologous bonds, although the former are the only likely ones. For a complex which is larger than a dimer, the only bonds possible between the subunit are heterologous bonds. (3) The proteins are glycoproteins in order to fix their transmembrane arrangement. This suggestion was made by Bretscher [24], and would be expected to apply to all eukaryotic membrane proteins which span the bilayer. If it turns out that transmembrane oligomerit glycoproteins are indeed required

TIBS - January I9 76

for transport across the membranes of eukaryotic cells, what about the properties of transport proteins in bacterial and mitochondrial membranes? There do not seem to be glycoproteins in the inner mitochondrial membrane and in the plasma membrane of most bacterial cells (but see the results of Mescher et al. [25]). Thus the glycosylation of transmembrane proteins may be a special feature of eukaryotic membranes. However, I suggest that bacterial membrane transport is catalyzed by oligomeric transmembrane proteins which resemble eukaryotic systems in all other properties. This suggestion is supported by recent evidence that bacterial rhodopsin, which is a light-activated proton pump, probably is a transmembrane protein which may be oligomeric [26]. References I Guidotti, G. (1972) Ann. Rev. Biochem. 41. 731. 752 2 Jorgensen, P.L. (1974) Biochim. Biophys. Acru 356, 53367 3 Clarke, S. (1975) J. Biof. Chem. 250, 5459-5469 4 Kyte, J. (1975) J. Biol. Chem. 250, 7443-7449 5 Jean, D.H.,Albers, R.W. and Koval, G.J. (1975) J. Biol. Chem. 250, 1035-1040

6 McLennan,

D.H. and Holland,

P.C. (1975) Ann.

Rev. BiophJ*s. Bioeng. 4, 377404

7 McLennan, D.H., Seeman, P., Iles, G.H. and Yip, C.C. (1971) J. Biol. Chem. 246.2707-2710 8 Warren, G.B.. Toon. P.A.. Birdsall, N.J.M ., Lee, A.G. and Metcalfe, J.C. (1974) Proc,. Nut. Acod. Sci. 71, 622-626 9 Bastide, F.. Meissner, G., Fleischer, S. and Post, R.L. (1973) J. Biol. Chem. 248,8385-8391 10 Gunn, R.B.. Dalmark, M., Tosteson, D.C. and Wieth. J.O. (1973) J. Gen. Physiol. 61, 185-206 II Ho. M.K. and Guidotti, G. (1975) J. Biol. Chem. 250, 675%683

12 Cabantchik, Membrane

Z.I.

and

Rothstein.

A. (1974)

J.

Biol. 15, 207-226

13 Wang. K. and Richards. F.M. (1974) J. Biol. C/rem. 249, 8005-8018 14 Bretscher, MS. (1971) J. Mol. Biol. 59, 351-357 I5 Karlin. A. (1974) Life Sci. 14. 138551415 I6 Martinez-Carrion, M., Sator, V. and Raftery, M.A. (1975) Biochem. Biophys. Res. Commun. 65, 1299137 I7 Karlin, A. and Cowburn. D. (1973) Proc. Nut. Acud. SC;. 70. 3636-3640 18 Michaelson, D.. Vandlen, R., Bode, J., Moody, T., Schmidt. J. and Raftery, M.A. (1974) Arch. Biochem. Biophys. 165, 7966804 I9 Hagins, W.A. (1972) Ann. Rev. Biophw. Bioeng. 1. 131-158 20 Lewis, M.S., Krieg, L.C. and Kirk, W.D. (1974) E.YP. Eye Res. 18, 2940 21 Chen. Y.S. and Hubbell. W.L. (1973) Esp. EJ,e Rex. 17, 517-532 22 Cone, R.A. (1975) in Functional Linkage in BioF.O., Schneider, molecular S.vstems (Schmitt, D.M. and Crothers, D.M., eds), pp. 234246. Raven Press, New York 23 Steinemann. A. and Stryer, L. (1973) Biochemis-

On the origin of primer for glycogen synthesis W. J. Whelan Glycogen and starch may be synthesized on a protein backbone, with the protein ,fi?rming un integral part of the macromolecular product.

The necessity for a ‘primer’ in polysaccharide synthesis was discovered by Carl and Gerty Cori. No sooner had they begun to purify their muscle-glycogen synthesizing system than it lost its ability to form glycogen from glucose l-phosphate [I]. The addition of a trace of glycogen, however, restored the capability. In making a similar observation in relation to starch synthesis in the potato, Hanes [2] concluded that the ‘activating’ effect of starch was caused by its chemical participation in the reaction. Cori et al. [3] confirmed this by the finding that ‘glycogen which had been added to prime the reaction could not be separated from the synthetic polysaccharide’. The primer is the polysaccharide itself, or a derived fragment containing the polymeric linkage, which may be as small as a di- or trisaccharide, and which the polymerase enzyme extends by adding monomer units to the non-reducing chain end of the primer. The expression ‘template’ has been used in place of primer, but the template in this case is not copied, as in nucleic acid synthesis, rather it is covalently incorporated into the product. The mechanism by which the primer itself is synthesized has often been debated. There may be no specific mechanism. Perhaps glycogen is ‘immortal’. One primer molecule can give rise to an infinity of primer molecules by chain elongation and hydrolytic fragmentation. Could every molecule of primer ever disappear? Condensation reactions of glucose, brought about by glycogen hydrolases, could lead to primer [4]. There is a report that glycogen synthase can add glucose from uridine diphosphate glucose (UDPG) to glucose, forming maltose [5]. However, in a spate of recent publications a specific mechanism for primer formation in glycogen and starch synthesis has seemingly been uncovered, and a new vista may have been opened on polysaccharide synthesis in general.

try 12.1499~1507

24 Bretscher, M.S. (1973) Science 181, 622-629 25 Mescher, M.F.. Strominger, J.L. and Watson, S.W. (1974) J. Bacrerioi. 945-954 26 Blaurock. A.E. (1975) J. Mol. Biol. 93, 139-158

Proteins as primers

The essentials of the enzyme system that W.J. W. is Professor Miumi

Schoolqf

Florida 33152,

of Biochemistry,

Medicine, USA

Universify

P.O. Box 520875,

of

Miami.

have been described in liver, potato and Escherichia coliare that glucose units from UDPG and/or adenosine diphosphate glucose (ADPG) are attached to an endogenous protein, resulting in products that resemble glycogen or starch and which, on account of their protein content, are precipitable by trichloroacetic acid. Krisman’s contributions, being among the earliest and most numerous, are cited in some detail [6]. A rat liver homogenate provides a 100 000-l 50 000 x g sediment which seems to contain three proteins (Fig. 1). One is a protein, or less likely, a glycoprotein (the protein primer), on which glycogen is built. The second protein (glycogen initiator synthase) is an enzyme that adds glucose units to the protein primer from UDPG, forming oligosaccharides that now act as classical primers and are elongated by the third protein, glycogen synthase, also utilizing UDPG. Despite these proteins being present as a mixture, their distinct properties permit their separate detection. The presence of the protein primer is made manifest by the fact that the labelled polysaccharide synthesized by the complete system is precipitable by trichloroacetic acid. After Pronase treatment, the product is trichloroacetic acidsoluble. If the enzyme system is complemented with soluble glycogen, polymerization of glucose from UDPG still occurs, but the product is not precipitable by acid. The exogenous glycogen competes as a primer with the endogenous protein primer. The presence of the second protein (glycogen initiator synthase) is shdwn’by its being necessary for the synthesis of acidprecipitable glycogen in the absence of added glycogen. This synthesis has a pH optimum of 8.7, and requires a high ionic strength medium, e.g. 100 mM ethylenediamine tetraacetate (EDTA) or ammonium sulphate. The presence of the third protein (glycogen synthase) is shown by the ability of the system, with added branching enzyme, to synthesize glycogen. The pH optimum of the synthase is 7.2. When the protein primer has been charged with glucose (from UDPG) in the high salt medium, further addition of glucose