Biology Reference
In-Depth Information
Vainberg,&Cowan, 1996
),which has precluded expression in bacterial systems.More-
over, unbalanced levels of
-subunits produce toxic effects in eukaryotes
(
Katz, Weinstein, & Solomon, 1990
), while overexpression hinders folding and pro-
duces nonfunctional aggregates (M. L. Gupta, unpublished results). Consequently,
the large-scale expression of exogenous tubulin has not been successful to date.
Conventionally,
in vitro
assays addressing microtubule function and regulation
by microtubule-associated proteins have largely utilized tubulin purified from mam-
malian brain tissue. This method yields large amounts of protein; however, the ma-
terial obtained is not homogeneous as the majority of higher eukaryotes express
multiple
a
-and
b
-tubulin isotypes. Additionally, site-specific mutagenesis of tubulin
expressed from the native locus is not possible or feasible in most higher eukaryotes.
Thus, purified brain tubulin is a heterogeneous mixture that confounds the analysis of
particular isotypes or mutations.
The budding yeast,
Saccharomyces cerevisiae
, is a powerful tool for systematic anal-
ysis of the functional consequences of tubulin mutations. Budding yeast encodes a single
b
a
- and
b
-tubulin isotypes,
TUB1
and
TUB3
(
Schatz, Pillus, Grisafi, Solomon, & Botstein, 1986
), which all share
-tubulin,
TUB2
(
Neff, Thomas, Grisafi, & Botstein, 1983
), and two
a
75% sequence conservation with human homologues. Furthermore, yeast can tolerate
deletion of
TUB3
, which can clarify
in vivo
readouts by eliminating influence of mul-
tiple isotypes and confer a source of homogeneous tubulin for
in vitro
analyses (
Bode,
Gupta, Suprenant, & Himes, 2003
). In budding yeast, site-specific mutations can be
inserted into the endogenous locus. Mutant proteins are expressed under the control of
the native promoter and regulatory elements, overcoming challenges and complexity
of mammalian systems. This approach eliminates difficulties associated with exogenous
expression and overexpression that may lead to nonphysiological results.
Budding yeast has been utilized for over 2 decades to study structural and
functional properties of tubulin and microtubules. Alanine-scanning mutagenesis
of
-tubulin uncovered clusters of charged amino acids crucial for structural
and functional integrity of tubulin and microtubules (
Reijo, Cooper, Beagle, &
Huffaker, 1994; Richards et al., 2000
). Directed mutagenesis demonstrated the
relationship between GTP hydrolysis and dynamic instability
in vivo
and
in vitro
(
Anders & Botstein, 2001; Davis, Sage, Dougherty, & Farrell, 1994
) and revealed
the contributions of microtubule dynamics to assembly and positioning of the mitotic
spindle (
Gupta et al., 2002; Huang & Huffaker, 2006
). Moreover, studies of yeast
tubulin have been important in defining binding sites of anticancer therapeutics
(
Gupta, Bode, Georg, & Himes, 2003
) and for discriminating among proposed
models for their interactions with microtubules (
Entwistle et al., 2012
). Recently,
structure-function analysis of yeast tubulin has been key for elucidating the molec-
ular etiology of a class of human neurological disorders that result from missense
mutations in distinct tubulin isotypes (
Cederquist et al., 2012; Jaglin et al., 2009;
Tischfield et al., 2010
).
Here, we present techniques to study the structural and functional consequences
of tubulin mutations using the budding yeast
S. cerevisiae
. This genetically tractable
system provides a well-controlled environment to relate the consequences of tubulin
a
- and
b
