Properties of microtubules assembled from mammalian tubulin synthesized in Escherichia coli.
When isolated from tissues, the alpha beta-dimeric protein tubulin consists of multiple isoforms which originate from the expression and subsequent posttranslational modification of multiple polypeptide sequences. Microtubules studied in vitro consist of mixtures of these isoforms. It is therefore not known whether dimers composed of single sequences of alpha- and beta-tubulin can polymerize to form microtubules, or whether posttranslational modifications may be necessary for microtubule assembly. To initiate investigation of these questions, rabbit reticulocyte lysate, which contains the cytoplasmic chaperonin CCT and its cofactors, was employed to prepare substantial quantities (tens of micrograms) of active tubulin by in vitro folding of mouse alpha- and beta-tubulins recombinantly synthesized in E. coli. This recombinant tubulin is composed of only a single alpha-chain and a single beta-chain. When analyzed after folding by isoelectric focusing, each chain yielded only one band, indicating that neither was detectably posttranslationally modified in the course of the folding reaction. When subjected to assembly-promoting conditions, this tubulin formed microtubules without the addition of any exogenous protein. Electron microscopy showed them to be of normal morphology. Analysis of their protein composition showed that they are composed nearly entirely of recombinant tubulin. These results demonstrate that the naturally occurring mixtures of isoforms are not strictly required for the formation of microtubules. They also open a route to other studies, both biomedical and structural, of fully defined tubulin in vitro.
Shah
C
C
Department of Biological Sciences, VU Station B 351634, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
Xu
C Z
CZ
Vickers
J
J
Williams
R
R
D000818:Animals; D002417:Cattle; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D007525:Isoelectric Focusing; D051379:Mice; D008870:Microtubules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D012996:Solutions; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D009124:Muscle Proteins; D011994:Recombinant Proteins; D012996:Solutions; D014404:Tubulin; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate