Science Magazine - 2 November 2012

interaction of Kap104 with this complex using
recombinant proteins derived from either C.
thermophilum or yeast (Fig. 4A and fig. S12E).
Size-exclusion chromatography revealed forma-
tion of a stable ctKap104-ctSyo1-ctRpl5-ctRpl11
import complex (Fig. 4A) with a 1:1:1:1 stoi-
chiometry of the four components, as determined
by static light scattering and refractive index mea-
surements (table S1). Addition of RanGTP was
sufficient to release the trimeric ctSyo1-ctRpl5-
ctRpl11 cargo complex from ctKap104 (Fig. 4B).
These findings indicate that Syo1 can serve as
the import adaptor for Rpl5-Rpl11 with Kap104
as the cognate import receptor for the hetero-
trimer. Given that Syo1 is not an essential pro-
tein, it is apparent that alternative import routes
for Rpl5 and Rpl11 must exist, which could in-
volve other transport receptors or diffusion across
the NPC.

A

120
140
M
peak fraction
B
GST-ctKap104

E
Syo1

NLS
AU (280 nm)

ctKap104
+
ctSyo1
ctRpl5
ctRpl11
kDa:
130
100
40
35
15
25
55
70
ctKap104
ctSyo1
Rpl5
Rpl11
ctRpl5
M
ctRpl11
20
50
0
0
C
kDa:
170
100

ct 5S-rRNA
100 150 200 250 300
Volume (ml)
M+
kDa:

GMPPNP
ctSyo1
ctRpl5-(His)6
ctRpl11
input
ctRan-
+
ct R an
tri m e ric c o m ple x
GST-
ctKap104
ctSyo1

Syo1-Rpl5-Rpl11
trimeric complex
Kap104

ct Rpl5
-(His)6
ct Ran
ctRpl11
Kap104-
Syo1-Rpl5-Rpl11
import complex
ctSyo1
15
1 23
45
ctRpl5-(His)6
cytoplasm
ctRpl11
15
D
AU (260 and 280 nm)
800
600
400
200
12
ctSyo1/ctRpl5/
ctRpl11/5S-rRNA
kDa:
130
70

NPC
nucleus

ctSyo1
ctRpl5
-(His)6
RanGTP
Kap104-
RanGTP

ctRpl11
release
M Ethidium bromide staining

130
70
35
25
15
55
Assembly of
Rpl5-Rpl11-5S
into pre-60S
ribosome
5S rRNA
5S-rRNA
0
pre-60S
6 8 10 12 14 16 18
Volume (ml)
Fig. 4. The Syo1-Rpl5-Rpl11 complex is imported into the nucleus via
Kap104 and binds to the 5S rRNA. (A) Size exclusion chromatography of the
reconstituted ctKap104-ctSyo1-ctRpl5-ctRpl11 heterotetrameric import com-
plex. The peak fraction was analyzed by SDS-PAGE and Coomassie staining. (B)
RanGMPPNP promotes release of the ctSyo1-ctRpl5-ctRpl11 complex from
ctKap104. The trimeric ctSyo1-ctRpl5-ctRpl11 complex (lane 4) was bound to
immobilized GST-ctKap104 (lane 1) and incubated in the absence (lane 2) or
presence (lane 3) of RanGMPPNP (lane 5). Input and bound proteins were
revealed by SDS-PAGE and Coomassie staining. M, molecular weight standard.
(C) Coomassie-stained SDS-PAGE of an in vitro binding assay of ctSyo1-
ctRpl5-ctRpl11 in the absence or presence of ct5S rRNA. The presence of 5S
rRNA did not lead to disassembly of the ctSyo1-ctRpl5-ctRpl11 complex. (D)
The ct5S rRNA and ctSyo1-ctRpl5-ctRpl11 form a stable complex on size ex-
clusion chromatography. Size exclusion chromatogram for free ct5S rRNA and
the ct5S rRNA-ctSyo1-ctRpl5-ctRpl11 complex at two different wavelengths
[i.e., 260 nm (red) and 280 nm (blue), upper panel]. SDS-PAGE of the peak
fractions stained with Coomassie (middle panel) and ethidium bromide
(lower panel) to detect the proteins and the 5S rRNA, respectively, present in
the ct5S rRNA-ctSyo1-ctRpl5-ctRpl11 complex. (E) The transport adaptor
Syo1 synchronizes nuclear import of Rpl5 and Rpl11. Syo1 simultaneously
interacts with Rpl5 and Rpl11 in the cytoplasm, thus enabling formation of a
tetrameric Kap104-Syo1-Rpl5-Rpl11 import complex. RanGTP binding to
Kap104 in the nucleus promotes release of the trimeric Syo1-Rpl5-Rpl11
complex, which recruits the 5S rRNA. Rpl5, Rpl11, and the 5S rRNA are then
incorporated with the aid of assembly factors (not shown) into pre-60S
ribosomes. See text for details.

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