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uncoating ATPase activation — Proposal of a new model
Medical Hypotheses
Hsc70Wncoating ATPase Activation - Proposal of a New Model 2. S. STANTCHEV Laboratory of Electron Microscopy, Varna institute of Medicine, Varna 9002, Bulgaria
Abstract - To the best of my knowledge, the signal for the clathrin removal from coated vesicles by the uncoating ATPase is still obscure. I presume that a specific configuration of preexisting, but separately ineffective uncoating ATPase binding sites, is responsible for the triggering of its action. Changes in the geometrical structure of clathrin that inevitably appear during the coated pit-coated vesicle conversion, place the preexisting uncoating ATPase recognition sites into the necessary disposition.
In my opinion, the question why only clathrin lattices of coated vesicles, but not that of coated pits, are disassembled by the 70kD heat shock protein (hsc70), designated also as uncoating ATPase, is still unsolved. The disassembly is ATP and clathrin light chain dependent (1,2). Recently, a model for triggering the action of hsc70 has been suggested (l), based on the following facts: Peptides encompassing residues 47-71 of the light chains bind hsc70 to stimulate ATP hydrolysis. The stimulatory effect is considerably greater for the peptide of clathrin light chain a (LCa) than for the peptide of clathrin light chain b (LCb). Changes in the local calcium or other ionic concentrations can induce reversible conformational change in the 47-71 residues region of LCa, but not in the same region of LCb. It has been postulated (1): the hsc70 binding site of LCa is cryptic in coated pits. Upon budding of a coated vesicle there is an ionic imbalance between the cytosol and the vesicle lumen. This results in transient ion fluxes across the vesicle membrane that cause conformational change in LCa, exposure of its hsc70 binding site and subsequent uncoating of the coated vesicle. Date received 20 October Date accepted 7 February
However, this model is not supported by some previous results showing (a) ion gradients across the vesicle membrane are not needed for uncoating of isolated coated vesicles (3), (b) changes in the calcium concentration within the physiological range insignificantly influence the rate of clathrin cages disassembly in vitro (3), and (c) cages containing exclusively LCa or LCb are equivalent as substrates for uncoating ATPase activity to native cages, where the two types of clathrin light changes are in random distribution (2). The formation of coated vesicles seems to be a constitutive process (4,5) and there is evidence for delivery of several unoccupied receptors via coated pits and vesicles to the peripheral endosoma1 compartment (6,7), i.e. uncoating of coated vesicles takes place without the possible ligand-induced changes in intracellular calcium (8,9) or concentrations of other intracellular ions (10,ll). In addition to calcium, clathrin light chains also bind calmodulin in a calcium dependent manner (12), but, to the best of my knowledge, there are no investigations dealing with the effect of calmodulin binding on the clathrin light chain-hsc70 interactions.
1993 1994
138
MEDICAL AUDIT NEWS
It has been found (a) hsc70 does not interact with light chain depleted cages (2), (b) uncombined clathrin light chains as well as free triskelions do not elicit ATP hydrolysis in the presence of hsc70 (13), and (c) preformed hsc70-ATP complexes interact with clathrin cages (2). It has been suggested that either light chains exist in a different conformation when bound to heavy chains, or sites on both heavy and light chains are needed for productive binding, or both (2). Probably, the peptides encompassing residues 47-7 1 have different conformation from the same regions in intact clathrin light chains, since they stimulate hsc70-mediated ATP hydrolysis. Using deep-etch electron microscopy, Heuser and Steer (14) have established (i) free clathrin triskelions bind up to three hsc70 molecules to their vertices in the absence of ATP, (ii) no hsc70 molecules can be found on disassembled triskelions when ATP is present - a fact supporting the notion that ATP controls an allosteric conversion of hsc70 between two different molecular configurations, and (iii) there is no evidence for any effect of hsc70 molecules on coated pits dynamics per se. Other observations have revealed that proteolytic removal of terminal domains appears to abolish the ability of clathrin cages to stimulate hsc70-mediated ATP hydrolysis and prevents cage dissociation (15). In my view, the above cited data do not suggest that the conversion of a coated pit into a coated vesicle leads to an exposure of a unique peptide loop recognized by hsc70 (1,16). Since hsc70 interacts with hydrophylic and charged peptides in vitro (17), its recognition sites may be present on the surface of clathrin cages rather than being exposed only during the disassembly process. I presume that the coated vesicle formation results in the creation of a specific, functionally active three-dimensional structure, consisting of more than one preexisting, but separately ineffective recognition sites for hsc70. The coated pit-coated vesicle conversion is inevitably connected with increase of the curvature of the clathrin lattice and transformation of some of its hexagons into pentagons (18). Upon these rearrangements the angles between some of the triskelions’ arms decrease and the distances between certain neighbouring light chains, as well as between certain terminal domains diminish (see Fig.). The shortened distances should facilitate the interaction of hsc70-ATP complexes with the necessary number of binding (activating) sites. If isolated 47-7 1 residues peptides are used, then there will be no limitations in their movement and binding to hsc70-ATP complexes. The model for hsc70 activation proposed herein suggests why coated pits do not lose their clathrin
139
i;) J
IXP
/’
“A
M
N
f
h Fig. (a) A schematic drawing of a clathrin triskelion. (b) A clathrin heavy chain. The distended end is designated as terminal domain. Three heavy chains noncovalently connect with their hydrophobic carboxyl termini to form the triskelion vertex. (c) A clathrin light chain. Two types of light chains have been identified, LCa and LCb. (d) A triskelion arm composed of one heavy and one noncovalently bound clathrin light chain. The two types of light chains are randomly distributed to clathrin heavy chains. The arms of different triskelions impose on and connect to each other to form the hexagons (f) and pentagons (g) of the clathrin coat (h). (e) A clathrin light chain and a part of a terminal domain in (f), (g) and (h). M, N, Ml, Nt: points marking the hsc70 recognition (binding) sites of clathrin light chains. Clathrin lattice consists of equilateral hexagons and pentagons with angles equal to 120” and 108” respectively. Triskelions are symmetrical structures, therefore AM=AN. As it is unlikely light chains to slide on heavy chains during pentagons formation, then AM=AN=AtMt=AtNt. c MAN=l20”, <: MtAtNt=l08” and AM=AtM)=AN=AtNt, therefore MtNr< MN. Analogous reasonings can be made for the distances between the neighbouring terminal domains in hexagons and pentagons,respectively.
coats - simply their curvature is too small. Probably hsc70-ATP complexes bind to clathrin at the latest steps of the coated pit-coated vesicle conversion. If increased calmodulin binding to clathrin light chains after ligand induced calcium influx (12,19) impedes their interactions with hsc70, then hsc70-ATP complexes should bind to the clathrin lattice of coated vesicles when calcium returns to resting values and calmodulin dissociates.
140
MEDICAL HYPOTHESES
In conclusion, I think that not ion induced changes in the conformation of LCa, but changes in the geometrical structure of the clathrin lattice as a whole are responsible for the uncoating ATPase activation. References I.
2.
3.
4.
5.
6.
7. 8.
Brodsky FM, Hill BL, Acton SL, Nathke DH. Ponnambalam S, Parham P. Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics, Trends Biochem Sci 1991; 16: 208-213. Schmid SL, Braell WA, Schlossman DM, Rothman JE. A role for clathrin light chains in the recognition of clathrin cages by ‘uncoating ATPase’. Nature 1984; 311: 228-231. Schlossman DM, Schmid SL, Braell WA, Rothman JE. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol 1984; 99: 723-733. Steinman RM, Mellman IS, Muller WA, Cohn ZA. Endocytosis and recycling of plasma membrane. J Cell Biol 1983; 96: I-27. O’Halloran TJ, Anderson RJW. Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium. J Cell Biol 1992; 118: 13711377. Basu SK, Goldstein JL, Anderson RJW, Brown MS. Monensin interrupts the recycling of low density lipo-protein receptors in human fibroblasts. Cell 1981; 24: 493-502. Watts C. Rapid endocytosis of the transferrin receptor in the absence of bound transferrin. J Cell Biol 198.5; 100: 663637. Rasmussen HJ, Barret PQ. Mechanism of action of Ca2+ - dependent hormones. In: Hormones and their Actions, Part II. In: Cooke BA, King RJB, van der Molen HJ eds. Elsevier Sci,
1988: 93-l 1I. 9 Block LH, Pletscher A. Receptor-mediated
10.
II.
12.
13.
14.
15.
16. 17.
18. 19.
Ca*+entry: diversity of function and mechanism. Trends Pharmacol Sci 1989; 9: 214-216. Ives HE, Rector FC, Jr. Proton transport and cell function. J Clin Invest 1984; 73: 285-290. Albers RW, Siegel GJ, Stahl WL. Membrane transport. In: Siegel et al, eds. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. (eds) 4th edn. Raven Press 1989: 4970. Lisanti MP, Shapiro LS. Moskowitz N, Hua EL, Puszkin S, Schook W. Isolation and preliminary characterization of clathrin associated proteins. Eon J Biochem 1982; 125: 463470. Braell WA, Schlossman DM, Schmid SL, Rothman JE. Dissociation of clathrin coats coupled to the hydrolysis at ATP: role of an uncoating ATPase. J Cell Biol 1984; 99: 73474 1, Heuser J, Steer CJ. Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction. J Cell Biol 1989; 109: 1457-1466. Schmid SL, Rothman JE. Two classes of binding sites for uncoating protein in clathrin triskelions. J Biol Chem 1985; 260: 10050-10056. Keen JH. Clathrin and associated assembly and disassembly proteins. Annu Rev Biochem 1990; 59: 415438. Flynn CC, Chappel TG, Rothman JE. Peptide binding and release by proteins implicated as catalysists of protein assembly. Science 1989; 245: 385-390. Heuser JE. Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol 1980; 84: 560-583. Salisbury JL, Condeelis JS, Maihle NJ, Satir P. Calmodulin localization during capping and receptor-mediated endocytosis. Nature 1981; 294: 163-166.
Hsc70Wncoating ATPase Activation - Proposal of a New Model 2. S. STANTCHEV Laboratory of Electron Microscopy, Varna institute of Medicine, Varna 9002, Bulgaria
Abstract - To the best of my knowledge, the signal for the clathrin removal from coated vesicles by the uncoating ATPase is still obscure. I presume that a specific configuration of preexisting, but separately ineffective uncoating ATPase binding sites, is responsible for the triggering of its action. Changes in the geometrical structure of clathrin that inevitably appear during the coated pit-coated vesicle conversion, place the preexisting uncoating ATPase recognition sites into the necessary disposition.
In my opinion, the question why only clathrin lattices of coated vesicles, but not that of coated pits, are disassembled by the 70kD heat shock protein (hsc70), designated also as uncoating ATPase, is still unsolved. The disassembly is ATP and clathrin light chain dependent (1,2). Recently, a model for triggering the action of hsc70 has been suggested (l), based on the following facts: Peptides encompassing residues 47-71 of the light chains bind hsc70 to stimulate ATP hydrolysis. The stimulatory effect is considerably greater for the peptide of clathrin light chain a (LCa) than for the peptide of clathrin light chain b (LCb). Changes in the local calcium or other ionic concentrations can induce reversible conformational change in the 47-71 residues region of LCa, but not in the same region of LCb. It has been postulated (1): the hsc70 binding site of LCa is cryptic in coated pits. Upon budding of a coated vesicle there is an ionic imbalance between the cytosol and the vesicle lumen. This results in transient ion fluxes across the vesicle membrane that cause conformational change in LCa, exposure of its hsc70 binding site and subsequent uncoating of the coated vesicle. Date received 20 October Date accepted 7 February
However, this model is not supported by some previous results showing (a) ion gradients across the vesicle membrane are not needed for uncoating of isolated coated vesicles (3), (b) changes in the calcium concentration within the physiological range insignificantly influence the rate of clathrin cages disassembly in vitro (3), and (c) cages containing exclusively LCa or LCb are equivalent as substrates for uncoating ATPase activity to native cages, where the two types of clathrin light changes are in random distribution (2). The formation of coated vesicles seems to be a constitutive process (4,5) and there is evidence for delivery of several unoccupied receptors via coated pits and vesicles to the peripheral endosoma1 compartment (6,7), i.e. uncoating of coated vesicles takes place without the possible ligand-induced changes in intracellular calcium (8,9) or concentrations of other intracellular ions (10,ll). In addition to calcium, clathrin light chains also bind calmodulin in a calcium dependent manner (12), but, to the best of my knowledge, there are no investigations dealing with the effect of calmodulin binding on the clathrin light chain-hsc70 interactions.
1993 1994
138
MEDICAL AUDIT NEWS
It has been found (a) hsc70 does not interact with light chain depleted cages (2), (b) uncombined clathrin light chains as well as free triskelions do not elicit ATP hydrolysis in the presence of hsc70 (13), and (c) preformed hsc70-ATP complexes interact with clathrin cages (2). It has been suggested that either light chains exist in a different conformation when bound to heavy chains, or sites on both heavy and light chains are needed for productive binding, or both (2). Probably, the peptides encompassing residues 47-7 1 have different conformation from the same regions in intact clathrin light chains, since they stimulate hsc70-mediated ATP hydrolysis. Using deep-etch electron microscopy, Heuser and Steer (14) have established (i) free clathrin triskelions bind up to three hsc70 molecules to their vertices in the absence of ATP, (ii) no hsc70 molecules can be found on disassembled triskelions when ATP is present - a fact supporting the notion that ATP controls an allosteric conversion of hsc70 between two different molecular configurations, and (iii) there is no evidence for any effect of hsc70 molecules on coated pits dynamics per se. Other observations have revealed that proteolytic removal of terminal domains appears to abolish the ability of clathrin cages to stimulate hsc70-mediated ATP hydrolysis and prevents cage dissociation (15). In my view, the above cited data do not suggest that the conversion of a coated pit into a coated vesicle leads to an exposure of a unique peptide loop recognized by hsc70 (1,16). Since hsc70 interacts with hydrophylic and charged peptides in vitro (17), its recognition sites may be present on the surface of clathrin cages rather than being exposed only during the disassembly process. I presume that the coated vesicle formation results in the creation of a specific, functionally active three-dimensional structure, consisting of more than one preexisting, but separately ineffective recognition sites for hsc70. The coated pit-coated vesicle conversion is inevitably connected with increase of the curvature of the clathrin lattice and transformation of some of its hexagons into pentagons (18). Upon these rearrangements the angles between some of the triskelions’ arms decrease and the distances between certain neighbouring light chains, as well as between certain terminal domains diminish (see Fig.). The shortened distances should facilitate the interaction of hsc70-ATP complexes with the necessary number of binding (activating) sites. If isolated 47-7 1 residues peptides are used, then there will be no limitations in their movement and binding to hsc70-ATP complexes. The model for hsc70 activation proposed herein suggests why coated pits do not lose their clathrin
139
i;) J
IXP
/’
“A
M
N
f
h Fig. (a) A schematic drawing of a clathrin triskelion. (b) A clathrin heavy chain. The distended end is designated as terminal domain. Three heavy chains noncovalently connect with their hydrophobic carboxyl termini to form the triskelion vertex. (c) A clathrin light chain. Two types of light chains have been identified, LCa and LCb. (d) A triskelion arm composed of one heavy and one noncovalently bound clathrin light chain. The two types of light chains are randomly distributed to clathrin heavy chains. The arms of different triskelions impose on and connect to each other to form the hexagons (f) and pentagons (g) of the clathrin coat (h). (e) A clathrin light chain and a part of a terminal domain in (f), (g) and (h). M, N, Ml, Nt: points marking the hsc70 recognition (binding) sites of clathrin light chains. Clathrin lattice consists of equilateral hexagons and pentagons with angles equal to 120” and 108” respectively. Triskelions are symmetrical structures, therefore AM=AN. As it is unlikely light chains to slide on heavy chains during pentagons formation, then AM=AN=AtMt=AtNt. c MAN=l20”, <: MtAtNt=l08” and AM=AtM)=AN=AtNt, therefore MtNr< MN. Analogous reasonings can be made for the distances between the neighbouring terminal domains in hexagons and pentagons,respectively.
coats - simply their curvature is too small. Probably hsc70-ATP complexes bind to clathrin at the latest steps of the coated pit-coated vesicle conversion. If increased calmodulin binding to clathrin light chains after ligand induced calcium influx (12,19) impedes their interactions with hsc70, then hsc70-ATP complexes should bind to the clathrin lattice of coated vesicles when calcium returns to resting values and calmodulin dissociates.
140
MEDICAL HYPOTHESES
In conclusion, I think that not ion induced changes in the conformation of LCa, but changes in the geometrical structure of the clathrin lattice as a whole are responsible for the uncoating ATPase activation. References I.
2.
3.
4.
5.
6.
7. 8.
Brodsky FM, Hill BL, Acton SL, Nathke DH. Ponnambalam S, Parham P. Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics, Trends Biochem Sci 1991; 16: 208-213. Schmid SL, Braell WA, Schlossman DM, Rothman JE. A role for clathrin light chains in the recognition of clathrin cages by ‘uncoating ATPase’. Nature 1984; 311: 228-231. Schlossman DM, Schmid SL, Braell WA, Rothman JE. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol 1984; 99: 723-733. Steinman RM, Mellman IS, Muller WA, Cohn ZA. Endocytosis and recycling of plasma membrane. J Cell Biol 1983; 96: I-27. O’Halloran TJ, Anderson RJW. Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium. J Cell Biol 1992; 118: 13711377. Basu SK, Goldstein JL, Anderson RJW, Brown MS. Monensin interrupts the recycling of low density lipo-protein receptors in human fibroblasts. Cell 1981; 24: 493-502. Watts C. Rapid endocytosis of the transferrin receptor in the absence of bound transferrin. J Cell Biol 198.5; 100: 663637. Rasmussen HJ, Barret PQ. Mechanism of action of Ca2+ - dependent hormones. In: Hormones and their Actions, Part II. In: Cooke BA, King RJB, van der Molen HJ eds. Elsevier Sci,
1988: 93-l 1I. 9 Block LH, Pletscher A. Receptor-mediated
10.
II.
12.
13.
14.
15.
16. 17.
18. 19.
Ca*+entry: diversity of function and mechanism. Trends Pharmacol Sci 1989; 9: 214-216. Ives HE, Rector FC, Jr. Proton transport and cell function. J Clin Invest 1984; 73: 285-290. Albers RW, Siegel GJ, Stahl WL. Membrane transport. In: Siegel et al, eds. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. (eds) 4th edn. Raven Press 1989: 4970. Lisanti MP, Shapiro LS. Moskowitz N, Hua EL, Puszkin S, Schook W. Isolation and preliminary characterization of clathrin associated proteins. Eon J Biochem 1982; 125: 463470. Braell WA, Schlossman DM, Schmid SL, Rothman JE. Dissociation of clathrin coats coupled to the hydrolysis at ATP: role of an uncoating ATPase. J Cell Biol 1984; 99: 73474 1, Heuser J, Steer CJ. Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction. J Cell Biol 1989; 109: 1457-1466. Schmid SL, Rothman JE. Two classes of binding sites for uncoating protein in clathrin triskelions. J Biol Chem 1985; 260: 10050-10056. Keen JH. Clathrin and associated assembly and disassembly proteins. Annu Rev Biochem 1990; 59: 415438. Flynn CC, Chappel TG, Rothman JE. Peptide binding and release by proteins implicated as catalysists of protein assembly. Science 1989; 245: 385-390. Heuser JE. Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol 1980; 84: 560-583. Salisbury JL, Condeelis JS, Maihle NJ, Satir P. Calmodulin localization during capping and receptor-mediated endocytosis. Nature 1981; 294: 163-166.