Participation of guanine nucleotides in nucleation and elongation steps of microtubule assembly.

Karr, Timothy L.; Podrasky, Ann E.; Purich, Daniel L.

doi:10.1073/pnas.76.11.5475

Cited by 56 publications

(32 citation statements)

References 19 publications

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“…It was subsequently reported that GDP would permit the elongation of microtubule seeds, but not de novo polymerization (Carlier & Pantaloni, 1978;Karr et al, 1979). It was therefore suggested that nucleation required a triphosphate in the exchangeable site.…”

Section: Discussionmentioning

confidence: 99%

Microtubule assembly with the guanosine 5'-diphosphate analog 2',3'-dideoxyguanosine 5'-diphosphate

Hamel¹,

Campo²,

Lin³

1983

Biochemistry

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The GDP analogue 2',2'-dideoxyguanosine 5'-diphosphate (ddGDP) supports efficient tubulin polymerization. Microtubule-associated protein (MAP) dependent microtubule assembly occurs in 0.1 M 2-(N-morpholino)-ethanesulfonate, and sheets of parallel protofilaments are formed in 1.0 M glutamate without MAPs. The nucleotide is bound to tubulin in the course of polymerization, presumably in the exchangeable GTP site. The ddGDP is not hydrolyzed, however, and is completely stable in the reaction mixture. Nor was the nonexchangeable GTP bound to tubulin hydrolyzed in ddGDP-supported polymerization: equivalent amounts of GTP remained associated with polymerized tubulin after polymerization with either ddGDP or GTP. Higher concentrations of ddGDP than GTP were required under all conditions. Nevertheless, under optimum conditions for the ddGDP-supported reaction, polymerization began with a shorter lag period and both the rate and extent of polymerization were greater with ddGDP than with GTP. The MAP-dependent reaction with ddGDP is temperature dependent, cold reversible, and inhibited by calcium and antimitotic drugs. It differs from the GTP-supported reaction in being most vigorous at minimal Mg2+ concentrations and exquisitely sensitive to GDP inhibition.

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Section: Discussionmentioning

confidence: 99%

Microtubule assembly with the guanosine 5'-diphosphate analog 2',3'-dideoxyguanosine 5'-diphosphate

Hamel¹,

Campo²,

Lin³

1983

Biochemistry

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“…(7) What is the mechanism for regulation of adenylate cyclase by Mg2+ (Cech et al, 1981) and other divalent cations and for activation of the adenylate cyclase system by NaF? (8) Finally, does the ubiquitous association of GTP-binding proteins with the regulatory function of GTP hydrolysis (Caskey et al, 1972;Karr et al, 1979;Shinozawa et al, 1979) suggest that the adenylate-cyclase-coupled G-protein may represent a macromolecular cue that synchronizes hormonestimulation with other, apparently unrelated, cellular functions?…”

Section: Summary Offuture Questionsmentioning

confidence: 99%

Activation and attenuation of adenylate cyclase. The role of GTP-binding proteins as macromolecular messengers in receptor–cyclase coupling

Limbird¹

1981

Biochemical Journal

321

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“…GTP binds rapidly and reversably to one exchangeable binding site (E-site) per tubulin subunit (1). During microtubule assembly, the E-site GTP undergoes a hydrolysis step as the subunit adds (2-5); and the GDP that is formed remains tightly bound, nearly one per dimer, in the polymer (3,4,6 (7)(8)(9)(10) and, reportedly, GDP (10)], the necessity for a hydrolysis step during assembly has been unclear.Microtubules undergo a constant opposite-end assembly/ disassembly mechanism in vitro (6). At an apparent equilibrium state for the polymer, subunits add at one end [the net assembly or (A) end] at a rate that is greater than their rate of loss at this end.…”

mentioning

confidence: 99%

“…Because nucleotides that are incapable of 13- (7)(8)(9)(10) and, reportedly, GDP (10)], the necessity for a hydrolysis step during assembly has been unclear.…”

mentioning

confidence: 99%

Role of GTP hydrolysis in microtubule treadmilling and assembly.

Margolis¹

1981

Proc. Natl. Acad. Sci. U.S.A.

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GTP hydrolysis accompanies addition of tubulin to microtubules. I find that hydrolysis is a requirement for the opposite-end assembly/disassembly of microtubules and consequent subunit treadmilling from one end to the other of the polymer. Neither GDP nor guanosine 5'-[fi,y-imido]triphosphate allows or participates in the treadmilling reaction. Therefore, there is a requirement for hydrolysis in the addition of subunits to the favored assembly end of the microtubule. Podophyllotoxin, an assembly inhibitory drug, "caps" the microtubule assembly end, preventing subunit loss from that site to equilibrium. Continued hydrolysis of GTP is required to maintain the podophyllotoxin cap. A corollary of this finding is that GTP hydrolysis is required for cap formation. Microtubules assembled in GTP enter a metastable state when all remaining GTP is hydrolyzed. This state is characterized by its ability to maintain indefinitely a subunit/polymer distribution ratio that is arbitrary and that can be altered at will by brief chilling or by addition of small amounts of GTP. This metastable state is labile to podophyllotoxin. Use of podophyllotoxin allows measurement of the microtubule treadmilling rate; use of podophyllotoxin in the absence of GTP allows measurement of the overall rate of dimer dissociation from the microtubule. Measurement of these rates has permitted determination of the efficiency with which adding dimers incorporate into the microtubule treadmill and are not lost to assembly end equilibrium. The efficiency varies with GTP concentration for unknown reasons, being high at 0.1 mM GTP and low at higher GTP concentrations. GTP binds rapidly and reversably to one exchangeable binding site (E-site) per tubulin subunit (1). During microtubule assembly, the E-site GTP undergoes a hydrolysis step as the subunit adds (2-5); and the GDP that is formed remains tightly bound, nearly one per dimer, in the polymer (3,4,6 (7)(8)(9)(10) and, reportedly, GDP (10)], the necessity for a hydrolysis step during assembly has been unclear.Microtubules undergo a constant opposite-end assembly/ disassembly mechanism in vitro (6). At an apparent equilibrium state for the polymer, subunits add at one end [the net assembly or (A) end] at a rate that is greater than their rate of loss at this end. As a consequence, the constant shuttling of subunits from one end to the other of a microtubule has been designated a "treadmilling" mechanism (11). A similar treadmilling mechanism occurs in actin polymers (12, 13). It has been suggested that ATP hydrolysis sustains the (A) end steady-state or treadmilling behavior in actin polymers (12).I report here that microtubules do not treadmill in the absence of GTP hydrolysis, although net assembly will occur in the presence of p[NH]ppG and a metastable state can be sustained in the presence of GDP. Furthermore, by examining the state of microtubules in the presence of GDP only, I find that hydrolysis creates stability.When microtubules are assembled with GTP and the GTP in solution is subs...

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Participation of guanine nucleotides in nucleation and elongation steps of microtubule assembly.

Abstract: Critical concentrations for formation of microtubules from subunits with GTP

Cited by 56 publications

References 19 publications

Microtubule assembly with the guanosine 5'-diphosphate analog 2',3'-dideoxyguanosine 5'-diphosphate

Microtubule assembly with the guanosine 5'-diphosphate analog 2',3'-dideoxyguanosine 5'-diphosphate

Activation and attenuation of adenylate cyclase. The role of GTP-binding proteins as macromolecular messengers in receptor–cyclase coupling

Role of GTP hydrolysis in microtubule treadmilling and assembly.

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