Biology Reference
In-Depth Information
The microtubule-severing enzymes katanin (
Hartman J.J. and Vale R.D. 1999)
and spastin (
Roll-Mecak & Vale, 2008
) assemble into biologically active hexamers
driven by ATP binding (
Fig. 13.1
B). The severing activity relies on the carboxy-
terminal tail (CTT) of tubulin (
Evans, Gomes, Reisenweber, Gundersen, &
Lauring, 2005; McNally & Vale, 1993; Roll-Mecak & Vale, 2005, 2008; White &
Lauring, 2007
). These enzymes are proposed to interact with the surface of the micro-
tubule lattice via an amino-terminal microtubule interacting and trafficking domain.
They also specifically bind to the CTT of tubulin via an active pore region of the
AAA
domains are thought to
translocate the CTT of the tubulin through the pore via ATP hydrolysis. This action
pulls the CTT of the tubulin subunit away from the lattice, possibly locally unfolding
the tubulin dimer, loosening the interdimer bonds, and generating a break in the
microtubule.
In vivo
, microtubule severing has been shown to be important for a vast number of
cellular processes, nevertheless, the location and regulation of severing enzymes
may vary depending on the subcellular region, cell type, and organism (recently
reviewed by
Sharp & Ross, 2012
).
þ
domain, the active site of the enzyme. The AAA
þ
Importance of in vitro biochemical and biophysical studies
Recapitulating a cellular process outside the cell greatly facilitates the development of
a mechanistic understanding of that process. The technique of
in vitro
reconstitution
enables one to control the components for a cellular process, with nothing superfluous,
to determine what is absolutely required for a given phenomenon to occur. Numerous
reductionist studies of this sort have been used over the past 30 years to learn much of
what we know about basic microtubule mechanics and dynamics. Using the methods
outlined in this chapter, we hope that more research can be conducted to illuminate the
underlying mechanisms controlling microtubule severing.
13.1
METHODS
Here, we present an itemized description of our procedure to purify and test
microtubule-severing enzymes
in vitro
. These methods were employed in our recent
publications (
Diaz-Valencia et al., 2011; Mukherjee et al., 2012b; Zhang et al.,
2011
).
FIGURE 13.1—Cont'd from the AAA domain of the three known severing enzymes. This
sequence shows conservation in all three enzymes of the important domains for the
enzyme function. The color scheme is the same as in B. (D) A structural representation of
the AAA region of spastin (PDB 3B9P). The zoomed-in views show the important residues
in the Walker A, Walker B, and pore loop regions. The color scheme is the same as in B.
