the very C-terminal part of the ctSyo1 disordered
loop (residues 395 to 408) showed a decrease
in deuterium incorporation upon ctRpl11 binding, indicating shielding of this ctSyo1 region
(fig. S6A). Consistent with these data, deletion
of residues 393 to 397 in the disordered loop
abolished binding to ctRpl11 (fig. S6, B and C).
Moreover, Y2H assays indicated that the ARM
domain of Syo1 contributes to Rpl11 binding
(fig. S7). From these results, we conclude that the
disordered loop of Syo1 in conjunction with the
ARM domain provides the interaction site for
Rpl11 (for a model, see fig. S8).
To act as import receptor, Syo1 must pass
through the hydrophobic FG-meshwork of the
nuclear pore complex (NPC) transport channel.
Shuttling karyopherin transport receptors do so
by binding with low (micromolar) affinity to FG
repeats of nucleoporins (12, 25). We found that
Syo1 and ctSyo1 bind to FG-rich repeats of dis-
tinct nucleoporins with a binding constant in the
low micromolar range (Fig. 3, A and B, and fig.
S9, A and B). Moreover, Syo1 can translocate
into the nucleus on its own in the absence of
cytosol, which provides karyopherin transport re-
ceptors and RanGTP, when tested in an in vitro
nuclear import assay with permeabilized HeLa
cells (26, 27) (Fig. 3C and fig. S9, C and D). In
agreement with translocation across the NPC
channel, Syo1 did not show nuclear accumula-
tion when permeabilized HeLa cells were in-
cubated with wheat germ agglutinin (WGA), a
specific inhibitor of nucleocytoplasmic transport
that sterically blocks the FG-repeat meshwork of
the NPC transport channel (Fig. 3E and fig. S9,
C and D). Thus, Syo1 can directly pass through the
NPC by low-affinity interaction with FG repeats
of NPC-channel nucleoporins.
transport receptor mediates nuclear import of
the Syo1-Rpl5-Rpl11 complex. Moreover, Syo1,
released from Rpl5 and Rpl11 after nuclear import, could shuttle back to the cytoplasm on its
own. In support of this possibility, Syo1 alone
traverses the NPC in both directions in permeabilized HeLa cells (Fig. 3 and fig. S9), and Syo1-
GFP (green fluorescent protein) is located both
in the nucleus and cytoplasm in yeast cells (fig.
S12, C and D).
To identify the import receptor of the Syo1-
Rpl5-Rpl11 complex, we took advantage of the
observation that low amounts of Kap104 were
sometimes coenriched when Syo1 was affinity-purified from yeast (fig. S11A). Kap104 is a member of the conserved karyopherin-b2/transportin
receptor subfamily (9), which recognizes a nonclassical NLS of the hydrophobic or basic proline-tyrosine (PY)–NLS type with the C-terminal
consensus signature R/K/H-X2–5-P-Y/L (29, 30)
(fig. S11B). Notably, Syo1 contains a conserved
putative basic PY-NLS at its N terminus. In vitro
binding assays revealed that this PY-NLS is necessary and sufficient for the interaction of Syo1
with Kap104 (fig. S11C and fig. S12A). Moreover, the PY-NLS can target an attached GFP
reporter into the nucleus in vivo and is required
for the in vivo function of Syo1 and nuclear
Fig. 2. Syo1 is structurally related to importins and binds the N-terminal
residues of Rpl5 in an extended groove. (A) Crystal structure of ctSyo1 (resi-
dues 25 to 675). Cartoon representation showing that ctSyo1 is an extended
a-solenoid, which consists of four complete ARM repeats (residues 65 to 260)
followed by six HEAT repeats (residues 274 to 675). The ARM and HEAT
repeats are indicated and “rainbow” colored from the N to the C terminus.
Dashed lines indicate disordered regions. (B) Surface representation of the
ctSyo1/ctL5-N complex. Residues 2 to 20 of ctRpl5 (L5-N, red) are accommo-
dated in an extensive groove of ctSyo1 (gray). (C) Multiple sequence align-
ment of residues 2 to 20 of L5-N from S. cerevisiae (Sc), Schizosaccharomyces
pombe (Sp), Homo sapiens (Hs), and C. thermophilum (Ct). Single-letter ab-
breviations for the amino acid residues are as follows: A, Ala; D, Asp; F, Phe;
G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S,
Ser; T, Thr; V, Val; and Y, Tyr. (D) Enlargement of the ctL5-N/ctSyo1 inter-
action. The 2Fobs – Fcalc electron density of ctL5-N at 1.3s after final refine-
ment is shown.