Supplementary Data

rapidcrimsonMechanics

Feb 22, 2014 (3 years and 7 months ago)

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Supplementary information


Crystal structure of

Cex1p reveals the mechanism of
tRNA trafficking between nucleus and cytoplasm


Kayo Nozawa, Ryuichiro Ishitani, Tohru Yoshihisa, Mamoru Sato, Naoshi
Dohmae, Dev Mangroo, Hubert D. Becker and Osamu Nureki


This document includes:

Supplementary Figure Legends



Suppl. Fig. 1

The c
rystal structures of
previously
the
reported

nuclear bound forms of
the
exportin

Los1p
from
Schizosaccharomyces pombe

(
a
)

and Exp
-
5

from
Homo sapiens

(
b
)
are shown

(both
includ
e

Gsp1p
)
. On the right

side
,
the

surface view
s

show the
electrostatic potential

(blue represents positive charge,
and

red negative

charge).

The

ribbon

models

on the left
show

the

equivalent views of Xpot and Exp
-
5
,
colored
in a
rainbow
.

In both complex structures,
the
tRNAs directly interact with
the
positively
charged HEAT repeat surface.


Suppl. Fig. 2

Structure
-
based s
equence alignment of
S.

cerevisiae

Cex1p

with
its
homologues
in
S. pombe
,
P
.

pastoris
,
A. thaliana
,
C. elegans
, D
. mel
anogaster

and
H. sapiens
.
White
letter
s

in r
ed boxes indicate conserved residues,

and

red letter
s

in white
boxes indicate
partially
conserved residues.

The kinase
-
like domain and
the
HEAT repeat domain of
Cex1p (1
-
544 aa)
are

conserved among the species
. B
lue
triangle
s
below the alignment
indicate amino

acids
constitut
ing
the
positively charged patch on the HEAT repeat
domain
, which was found to interact with tRNA
.


Suppl. Fig. 3

Cloverleaf structures of yeast
tRNA
Met

(a),

tRNA
Phe

(b) and tRNA
Phe

ASL

(c)
.
Y
east tRNA
Phe

contains

modified nucleosides, including
methylat
ed nucleosides

(indicated by “m”)
,

pseudouridine (Ψ), dihydrouridine (D) and wybutosine (yW).
The
y
east tRNA
Met

and
tRNA
Phe

ASL

used in this study
were
prepared by

in vitro

transcription

and
thu
s lack

base modifications.

The n
umbering of the bases in the
truncated tRNA
Phe

is based on the standard numbering of

yeast tRNA
Phe
.


Suppl. Fig.

4

We measured far
-
UV CD spectra of all Cex1p constructs. An α
-
helical structure
would give negative contributi
on at around 208 and 222 nm in the CD spectrum, are
observed for all Cex1p constructs. This result of Cex1p mutants is in good agreement
with the secondary structure content observed in crystal structure of Cex1pΔC
-
SER.


Suppl. Fig.

5

A
nalytical
ultracentrifugation experiments

on Cex1p constructs (
S
edimentation
velocity experiment
and
Sedimentation equilibrium experiments
).
The sedimentation
coefficient profiles
for

wild

type
Cex1p

(top),
Cex1pΔC
-
SER

(middle),
and
r
epresentative results of the sedim
entation equilibrium experiment

for
Cex1p9
-
358

(bottom)

are shown.
The absorbance at 280nm versus the radius and the best
-
fit curve
for
Cex1p9
-
358

are

shown in the bottom panel. The residuals of the fit from the
Cex1p9
-
358

data are shown in the upper panel
.

Those data are
correspond
ing to each
dimer molecular weight of Cex1p constructs.