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-
ct
Kap104
E
Syo1
NLS
AU (280 nm)
ct
Kap104
+
ct
Syo1
ct
Rpl5
ct
Rpl11
kDa:
130
100
40
35
15
25
55
70
ct
Kap104
ct
Syo1
Rpl5
Rpl11
ct
Rpl5
M
ct
Rpl11
20
50
0
0
C
kDa:
170
100
ct
5S-rRNA
100 150 200 250 300
Volume (ml)
M+
kDa:
GMPPNP
ct
Syo1
ct
Rpl5-(His)
6
ct
Rpl11
input
ct
Ran-
+
ct
R an
tri m e ric c o m ple x
GST-
ct
Kap104
ct
Syo1
Syo1-Rpl5-Rpl11
trimeric complex
Kap104
ct
Rpl5
-(His)
6
ct
Ran
ct
Rpl11
Kap104-
Syo1-Rpl5-Rpl11
import complex
ct
Syo1
15
1 23
45
ct
Rpl5-(His)
6
cytoplasm
ct
Rpl11
15
D
AU (260 and 280 nm)
800
600
400
200
12
ct
Syo1/
ct
Rpl5/
ct
Rpl11/5S-rRNA
kDa:
130
70
NPC
nucleus
ct
Syo1
ct
Rpl5
-(His)
6
RanGTP
Kap104-
RanGTP
ct
Rpl11
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.
Cover
IFC
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
IBC
BC
Zoom level
fit page
fit width
A
A
fullscreen
one page
two pages
print
SlideShow
fullscreen
in this issue
search
back issues
help
Open Article
Open Article
Close Article
article text for page
< previous story
|
next story >
Share this page with a friend
Save to “My Stuff”
Subscribe to this magazine
Search
Help