From the Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Received for publication, September 4, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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Little is known about the mechanisms that
organize the internal membrane systems in eukaryotic cells. We are
addressing this question in striated muscle, which contains two novel
systems of internal membranes, the transverse tubules and the
sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an ~17-kDa
transmembrane protein of the SR that concentrates around the Z-disks
and M-lines of each sarcomere. We used the yeast two-hybrid assay to
determine whether sAnk1 interacts with titin, a giant myofibrillar
protein that organizes the sarcomere. We found that the hydrophilic
cytoplasmic domain of sAnk1 interacted with the two most
N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the
Z-line in situ. Both ZIg1 and ZIg2 were required for
binding activity. sAnk1 did not interact with other sequences of titin
that span the Z-disk or with Ig domains of titin near the M-line. Titin
ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We
show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle
homogenates and suggest a role for these proteins in organizing the SR
around the contractile apparatus at the Z-line.
As striated muscle develops, the basic contractile unit, the
sarcomere, is assembled before the transverse tubules
(T-tubules)1 and the
sarcoplasmic reticulum (SR) mature (1, 2). The contractile cycle in
striated muscle normally requires the spread of the action potential
along the T-tubules into the interior of the muscle fiber, where
depolarization induces the release of Ca2+ from the
terminal cisternae of the SR, causing contraction (2, 3). Relaxation
follows the re-uptake of Ca2+ into a distinct domain of the
SR, which has been referred to as the longitudinal or network SR (4).
Typically, the network SR is positioned around the Z-disks and M-lines
of each sarcomere, but the structural elements that determine its
location have not been determined. Early ultrastructural studies
demonstrated the presence of numerous filaments joining the
periphery of sarcomeric Z-disks to adjacent SR membranes (5), but
the molecular identity of these structures remains elusive.
We have searched for protein partners of small ankyrin-1 (sAnk1), a
muscle-specific isoform of the erythroid ankyrin-1 gene that is
concentrated in the network SR of striated muscle fibers, surrounding
the Z-disks and M-lines (6). Ankyrins are a family of proteins that
possess binding sites for diverse integral membrane proteins as well as
cytoskeletal components (7-9). To date, molecular cloning has
identified three distinct ankyrin genes in mammals (Ank1,
Ank2, and Ank3) that are expressed as
tissue-specific, alternatively spliced isoforms (10-12).
Ank1 is expressed predominantly in erythroid cells,
striated muscle, and brain (13-15); Ank2 in brain and
cardiac muscle (16-19); and Ank3 in cells of epithelial origin and striated muscle as well as in lysosomes and Golgi membranes in a wide variety of cells (20-23). The large canonical ankyrins share
a similar structure, consisting of an N-terminal ~89-kDa membrane-binding domain, a central ~62-kDa spectrin-binding domain, and a C-terminal ~55-kDa regulatory domain (10, 11).
In striated muscle, the products of the Ank1 gene include
the large (~210 kDa) and small (~17-19 kDa) ankyrin isoforms (6, 15). sAnk1 lacks both the membrane- and spectrin-binding regions of the
larger form and has a C-terminal domain that is significantly shortened
(24, 25). The N-terminal portion of sAnk1 contains a unique 73-amino
acid segment, whereas the C-terminal 82 residues are identical to the
C-terminal sequence of the large ~210-kDa ankyrin-1. The first 29 residues of sAnk1 are highly hydrophobic and target the molecule to the
SR membrane, whereas the remaining 126 amino acids extend into the
myoplasm.2 Thus, the
hydrophilic tail of sAnk1 is appropriately oriented in the cytoplasm of
striated muscle fibers to serve as a binding site for sarcomeric proteins.
Here, we describe a direct and specific association between sAnk1 and
titin, also known as connectin (25-29). Titin is a giant (~2.7-4
MDa) protein that extends from the Z-disk to the M-line within the
sarcomere, which it helps to organize. It is highly modular: ~90% of
its mass is composed of repeating Ig-C2 and fibronectin-3-like domains that provide binding sites for myofibrillar proteins (31, 32).
The remaining ~10% consists of unique non-repetitive sequence motifs, including phosphorylation sites, binding sites for
muscle-specific calpain proteases, and C-terminal Ser/Thr kinase
domains (30, 33-35).
The C-terminal 2 MDa of titin are located within the A-band, where
titin tightly associates with the myosin thick filaments and several
A-band proteins such as C-protein, M-protein, and myomesin (36-38).
The most C-terminal end of the molecule (~200 kDa), which is embedded
in the M-line, contains a Ser/Thr kinase domain, which implicates titin
in myofibrillar signal transduction pathways (37-40). In the I-band,
titin (~800 kDa to 1.5 MDa) carries proline/glutamate/valine/lysine-rich sequences, which confer great extensibility to the titin filaments (35, 41-44), in addition to
numerous Ig domains. At the junction of the I-band with the Z-disk,
titin interacts with the actin thin filaments, although it is still
unclear which titin motifs mediate this interaction (45, 46). The
N-terminal 80-kDa region of titin spans the entire Z-disk (47). Several
copies of a 45-residue repeat, called the Z-repeat, bind Titin has two functions in striated muscle: as a "molecular
blueprint" for sarcomeric protein assembly during myofibrillogenesis and as a "molecular spring" that maintains myofibrillar integrity during contraction, relaxation, and stretch (27, 30, 32). Our results
show that, in addition to binding T-cap, the two N-terminal Ig domains
of titin interact specifically with sAnk1, suggesting that titin also
coordinates the assembly of the contractile apparatus with the network
SR that surrounds the Z-disk.
Generation of Titin, sAnk1, and T-cap Expression
Constructs--
PCR amplification was used to obtain cDNAs
encoding fragments of the Z-disk (~80 kDa) (47) and M-line (~200
kDa) (37) portions of titin. cDNA from human cardiac muscle
(Origene Technologies Inc., Rockville, MD) was used as
template, and the following sets of custom oligonucleotide primers were
generated, based on the sequence of human cardiac titin
(GenBankTM/EBI accession number X90568). For amplification
of ZIg1/2, the two most N-terminal Ig domains of titin, primers A
(5'-ACGTGAATTCATGACAACTCAAGCACCG-3', sense) and B
(5'-ACGTCTCGAGAGGTACTTCTTCTTCACC-3', antisense) were used. For ZIg1/2-A
(including ZIg1 only), the sense primer A was used in combination with
the antisense primer C (5'-ACGTCTCGAGAGCTTTCACGAGAAGCTC-3'). For
generation of ZIg1/2-B (containing ZIg2 only), primer D
(5'-ACGTGAATTCGAGACAGCACCACCCAAC-3') was used along with the
antisense primer B. For ZIg3, the Ig domain just C-terminal to ZIg1/2,
primer E (5'-ACGTGAATTCGCTAAAAAGACAAAGACA-3', sense) was utilized in
combination with primer F (5'-ACGTCTCGAGCATTATTGCTTCTTGAGT-3', antisense). For amplification of Zr (for
Z-repeat), the region of titin that interacts
with
Similarly, a fragment encoding the C-terminal hydrophilic sequence of
sAnk1-(29-155) (25) was inserted into the yeast two-hybrid pLexA bait vector and the pGEX4T-1 vector at
EcoRI/XhoI sites after PCR amplification with
primers 1 (5'-ACTGGAATTCGTCAAGGGTTCCCTGTGC-3', sense) and 2 (5'-ACTGCTCGAGCTGCTTGCCCCTTTT, antisense). An identical set of primers
carrying EcoRI and SalI recognition sites was
used for insertion into the pMAL-c2X vector to produce a
maltose-binding protein (MBP) fusion peptide. Additional sets of
primers were used for amplification of sAnk1 deletion constructs. For
sAnk1-A, the sense primer 1 was used in combination with the antisense primer 3 (5'-ACTGCTCGAGTTGTTCCTCTGTCAC-3'). For generation of sAnk1-B,
the sense primer 4 (5'-ACTGGAATTCTTCACAGACGAACAG-3') was used with the
antisense primer 2. For sAnk1-C, primer 5 (5'-ACTGGAATCATCTCCACCAGGGTG-3', sense) was used with primer 6 (5'-ACTGCTCGAGTCCACTCCCTCTTAG-3', antisense).
For generation of MBP-T-cap fusion protein, the full-length pET9D-T-cap
plasmid (a generous gift from Drs. S. Labeit (European Molecular
Biology Laboratory, Heidelberg, Germany) and C. C. Gregorio (University of Arizona, Tucson, AZ)) was used as template to obtain a
PCR fragment that contained amino acids 1-140 (47). Primers 7 (5'-ACGTGAATTCATGGCTACCTCAGAGCTG-3', sense) and 8 (5'-ACGTGTCGACTCATGTCTCCAGCGCCAG-3', antisense), carrying
EcoRI and SalI sites, respectively, were used for
insertion into the pMAL-c2X vector. T-cap-(1-140) was also introduced
into the pGEX4T-1 vector at EcoRI/XhoI sites
(XhoI and SalI have compatible ends) to produce a
GST fusion protein. The authenticity of the obtained constructs was
verified by sequencing analysis. GST and MBP recombinant
polypeptides were expressed by induction with 0.5 mM
isopropyl- Yeast Two-hybrid and
Liquid GST Pull-down Assay--
Homogenates of quadriceps muscle of
adult Sprague-Dawley rats (Zivic-Miller Laboratories, Inc.,
Zelienople, PA) were prepared at room temperature for 2-3 min
with a Brinkmann Polytron homogenizer at setting 3 (VWR, West Chester,
PA) in 10 mM NaPO4 (pH 7.2), 2 mM
EDTA, 10 mM NaN3, 120 mM NaCl, and
1% Nonidet P-40 supplemented with a mixture of protease inhibitors
(Roche Molecular Biochemicals). Equal amounts of recombinant GST and
GST-ZIg1/2, GST-Zr, GST-ZIg3, GST-ZIg4/5, GST-MIg1/2, and GST-MIg5/6
fusion proteins were bound to glutathione-Sepharose and mixed with 0.5 mg of quadriceps muscle homogenate at 4 °C for 16 h. Beads were
washed in the cold with 10 mM NaPO4 (pH 7.2),
120 mM NaCl, 10 mM NaN3, and 0.1%
Tween 20 and heated for 5 min at 90 °C in 2× SDS Laemmli sample
buffer. The soluble fraction was analyzed by 12% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to sAnk1. Immunoreactive bands were visualized with a chemiluminescence detection kit (Tropix Inc., Bedford, MA).
Blot Overlay--
The blot overlay assays were performed as
previously described with minor modifications (51). Briefly, ~2.5
µg of bacterially expressed, affinity-purified GST and GST-ZIg1/2
proteins were separated by 12% SDS-PAGE and transferred to
nitrocellulose. Nonspecific sites on the nitrocellulose membranes were
blocked in buffer A (50 mM Tris (pH 7.2), 120 mM NaCl, 3% bovine serum albumin, 2 mM
dithiothreitol, 0.5% Nonidet P-40, and 0.1% Tween 20) plus protease
inhibitors for 3 h at 25 °C and then incubated with 3 µg/ml
MBP-sAnk1 fusion protein in the same buffer for 16 h at 4 °C.
Blots were washed five times (15 min each) with buffer A and once with
buffer B (1× phosphate-buffered saline (pH 7.2), 10 mM
NaN3, and 0.1% Tween 20). Subsequently, they were
incubated in buffer C (1× phosphate-buffered saline (pH 7.2), 10 mM NaN3, 0.1% Tween 20, and 3% dry milk) and
probed with antibodies to sAnk1, diluted in buffer C. In a set of
parallel assays, increasing concentrations of affinity-purified
GST-ZIg1/2 fusion protein (i.e. 5 and 10 µg) were added to
buffer A along with MBP-sAnk1, and blots were processed as just described.
In Vitro "Competition" Assay--
Equivalent amounts of GST
protein, GST-sAnk1, and GST-T-cap bound to glutathione matrices were
allowed to interact with 5 µg of recombinant MBP-ZIg1/2, MBP-sAnk1,
or MBP-T-cap in 250 µl of binding buffer (50 mM Tris (pH
7.2), 120 mM NaCl, 10 mM NaN3, 2 mM dithiothreitol, 0.1% Tween 20, and 10 mM
maltose plus protease inhibitors) for 12 h at 4 °C.
Subsequently, the supernatants were removed, and the glutathione beads
were extensively washed with a solution containing 50 mM
Tris (pH 7.2), 120 mM NaCl, 10 mM NaN3, and 0.1% Tween 20. In a parallel set of experiments,
GST-sAnk1 and GST-T-cap attached to glutathione matrices were initially allowed to interact with 5 µg of bacterially expressed MBP-ZIg1/2 for
12 h at 4 °C. Following removal of the supernatants, 5 µg of
affinity-purified MBP-T-cap or MBP-sAnk1 were added to the GST-sAnk1·MBP-ZIg1/2 or GST-T-cap·MBP-ZIg1/2 complexes,
respectively, and allowed to interact for another 12 h in the
cold. At the end of the incubation period, the glutathione beads were
washed four times (15 min each) and dissolved in 2× SDS Laemmli sample
buffer. The soluble proteins were fractionated on 12% SDS-PAGE,
transferred to nitrocellulose, and probed with the appropriate antibodies.
Immunofluorescent Labeling and Confocal Microscopy Studies of
Adult Skeletal Muscle--
Frozen longitudinal sections and
cross-sections of quadriceps muscle of adult rats were prepared as
previously described (52). Sections were incubated in buffer D (1×
phosphate-buffered saline, 5% normal donkey serum, and 10 mM NaN3) for 1-2 h at 25 °C. Primary antibodies, including rabbit anti-titin-x112/x113 (3 µg/ml; a generous gift from Dr. C. C. Gregorio), rabbit anti-sAnk1 (3 µg/ml) (6), goat anti-telethonin (N-20 or C-20, 6 µg/ml; Santa Cruz Biotechnology), and rabbit (3 µg/ml) or goat (6 µg/ml) ChromaPure IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), were
diluted in buffer D and added to the sections for 12 h at 4 °C.
Following extensive washing with the same buffer, samples were
counterstained with the appropriate secondary antibodies, including
Alexa-568 goat anti-rabbit IgG and Alexa-488 donkey anti-goat
IgG (Molecular Probes, Inc., Eugene, OR), in buffer D at
1:100 dilution for 1 h at 25 °C. Subsequently, sections were washed three times (15 min each), mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA), and analyzed with a Zeiss 410 confocal laser scanning microscope (Carl Zeiss, Inc., Tarrytown, NY)
equipped with a ×63 NA 1.4 objective.
Materials--
Unless otherwise noted, all reagents were from
Sigma and were the highest grade available.
Binding of sAnk1 to the Two Most N-terminal Ig Domains of Titin
Shown with the Yeast Two-hybrid Assay--
sAnk1 is an ~17-19-kDa
integral membrane protein of the network SR that has a 126-amino acid
sequence extending into the sarcoplasm surrounding Z-disks and M-lines
(6).2 We used the yeast two-hybrid assay to test the idea
that the hydrophilic sequence of sAnk1 (sAnk1-(29-155)) interacts with the giant myofibrillar protein titin, which spans each half-sarcomere from the Z-disk to the M-line. We inserted cDNA encoding the
hydrophilic cytoplasmic domain of sAnk1 (sAnk1-(29-155)) (25) into the
yeast two-hybrid pLexA bait vector (Fig.
1A) and assayed its ability to
interact with the N-terminal ~80-kDa portion of titin that resides in
the Z-line (47), expressed by a series of constructs inserted into the
yeast two-hybrid pB42AD prey vector (Fig. 1B). Specifically,
the PCR products of titin we assayed were ZIg1/2 (amino acids 1-200),
ZIg3 (amino acids 201-557), the Z-repeats or Zr domain (amino acids
558-910), and ZIg4/5 (amino acids 911-1118) (see "Experimental
Procedures"). Yeast two-hybrid analysis followed by qualitative
liquid
In additional tests of the specificity of the interaction with ZIg1/2,
we generated two additional "prey" constructs encoding tandem Ig
domains that reside in the M-line region of titin (37-39). These included MIg1/2 (amino acids 25250-25422) and MIg5/6 (amino acids 26281-26478). When their ability to interact with
sAnk1-(29-155) was tested in a yeast two-hybrid assay, no specific
interaction was observed (Fig. 1C). Although we were unable
to test the ability of other M-line domains of titin to interact with
sAnk1 in this assay, our results suggest that sAnk1 interacts
preferentially with Ig domains at the N-terminal region of titin,
located at the Z-disk.
Mapping the Binding Sites on sAnk1 and Titin
ZIg1/2--
We used the yeast two-hybrid assay to identify
more precisely the sequences required for the binding of sAnk1 to titin
(Fig. 2). Three subfragments of the
hydrophilic portion of sAnk1 were inserted into the pLexA bait vector:
sAnk1-A (amino acids 29-89), sAnk1-B (amino acids 90-155), and
sAnk1-C (amino acids 61-130) (Fig. 2A). Two
truncated titin ZIg1/2 prey constructs, ZIg1 (amino acids 1-99) and
ZIg2 (amino acids 100-200), were also generated to assay the ability
of each of the N-terminal Ig domains of titin to bind sAnk1
independently (Fig. 2B). Yeast two-hybrid analysis followed
by liquid Binding of sAnk1 in Muscle Homogenates to GST-ZIg1/2 in
a Pull-down Assay--
To confirm the specificity of the interaction
between sAnk1 and titin ZIg1/2, we performed a GST pull-down assay
using homogenates of skeletal muscle from adult rats. We expressed
ZIg1/2, ZIg3, Zr, ZIg4/5, MIg1/2, and MIg5/6 as GST fusion proteins
(Fig. 3). The calculated molecular masses
of the GST fusion proteins are ~47, ~65, ~64, ~48, ~44, and
~47 kDa, respectively (Fig. 3A). GST-ZIg3 and GST-Zr
showed some degradation, probably due to endogenous bacterial
proteases, whereas GST-ZIg4/5 migrated with an apparent molecular mass
of ~60 kDa instead of the calculated ~48 kDa. Equivalent amounts of
these proteins and control GST (25 kDa) were bound to glutathione
matrices and incubated with homogenates of quadriceps muscle. The
matrix-bound GST fusion proteins were examined by Western blotting for
their ability to adsorb native sAnk1. Only GST-ZIg1/2 specifically
retained native sAnk1 (Fig. 3B). None of the other Z-disk or
M-line fragments of titin that we examined adsorbed sAnk1 from muscle
homogenates, consistent with the yeast two-hybrid data (Fig.
1C). These results indicate that the two most N-terminal Ig
domains of titin can bind to sAnk1 in muscle homogenates, consistent
with the association of sAnk1 with this region of titin in
vivo.
To determine whether sAnk1 binds titin ZIg1/2 directly, we
performed an overlay assay with bacterially expressed
MBP-sAnk1-(29-155) (~56 kDa) and GST-ZIg1/2 (~47 kDa) fusion
proteins (Fig. 4A). Equivalent
amounts of GST-ZIg1/2 and control GST were subjected to SDS-PAGE,
transferred to nitrocellulose membranes, and overlaid with
affinity-purified MBP-sAnk1-(29-155). Recombinant sAnk1 specifically bound to GST-ZIg1/2 fusion protein, but not to control GST protein, as
shown by immunoblotting with antibodies to sAnk1 (Fig. 4B). The ability of recombinant sAnk1 to bind GST-ZIg1/2 directly was inhibited by including soluble GST-ZIg1/2 (5 and 10 µg) in the overlay buffer along with MBP-sAnk1-(29-155) (Fig. 4B),
confirming the specificity of the direct interaction between sAnk1 and
titin ZIg1/2.
sAnk1 and T-cap Bind to Titin ZIg1/2
Simultaneously--
The two most N-terminal Ig domains of titin were
shown previously to bind to a striated muscle-specific, 19-kDa Z-disk
protein named T-cap or telethonin (47, 50). Our yeast two-hybrid and in vitro binding studies indicated that titin ZIg1/2 also
bound to a 29-amino acid segment (i.e. amino acids
61-89) within the hydrophilic segment of sAnk1. To determine whether
titin ZIg1/2 can bind simultaneously to sAnk1 and T-cap or whether
binding to one abolishes binding to the other, we performed an in
vitro competition assay using bacterially expressed sAnk1,
T-cap, and titin ZIg1/2 proteins.
We generated the following fusion proteins for these experiments:
GST-sAnk1-(29-155) (~39 kDa), MBP-sAnk1-(29-155) (~56 kDa), GST-T-cap-(1-140) (~40 kDa), MBP-T-cap-(1-140) (~57 kDa), and MBP-ZIg1/2 (~64 kDa) (Fig.
5A). We included the
N-terminal 1-140 residues (~16 kDa) of T-cap in the fusion protein,
as they contain the binding site for titin ZIg1/2 (47, 50). Equivalent
amounts of GST-sAnk1, GST-T-cap, and GST protein bound to glutathione matrices were incubated with MBP-ZIg1/2. Both GST-sAnk1 and GST-T-cap specifically retained recombinant titin ZIg1/2, whereas control GST
protein did not, as shown by Western blot analysis with anti-titin ZIg1/2 antibody (Fig. 5B). In a set of parallel assays,
GST-sAnk1 and GST-T-cap attached to glutathione beads were initially
allowed to interact with MBP-ZIg1/2. Following removal of unbound
recombinant titin ZIg1/2, affinity-purified MBP-T-cap and MBP-sAnk1
were added to GST-sAnk1·MBP-ZIg1/2 and GST-T-cap·MBP-ZIg1/2
complexes, respectively, or to control matrix-bound GST protein. The
presence of MBP-ZIg1/2, MBP-T-cap, and MBP-sAnk1 was examined in each
sample by immunoblot analysis with the appropriate antibodies
(i.e. anti-titin ZIg1/2, anti-T-cap, and anti-sAnk1) (Fig.
5, B-D). Both recombinant sAnk1 (Fig. 5C) and
T-cap (Fig. 5D) bound efficiently and specifically to
GST-T-cap·MBP-ZIg1/2 and GST-sAnk1·MBP-ZIg1/2 complexes,
respectively. To rule out the possibility of a direct association
between sAnk1 and T-cap, equivalent amounts of GST-sAnk1 and GST-T-cap
adsorbed to glutathione matrices were also incubated with
affinity-purified MBP-T-cap and MBP-sAnk1, respectively, under the same
experimental conditions. No association was observed between the two
proteins (data not shown). Our results indicate that the two N-terminal Ig domains of titin (ZIg1/2) simultaneously bind sAnk1 and T-cap in vitro, forming a three-way complex.
Subcellular Distribution of sAnk1, Titin ZIg1/2, and
T-cap in Adult Skeletal Muscle Fibers--
It has been well documented
that sAnk1, titin ZIg1/2, and T-cap are present at the Z-lines of
sarcomeres (6, 47, 54). To examine their topography with respect to the
Z-disk, we used antibodies against sAnk1, titin ZIg1/2, and T-cap to
label longitudinal sections and cross-sections of muscle fibers by
immunofluorescence, followed by confocal microscopy. As expected, each
of the antibodies labeled the Z-lines in longitudinal sections of adult
rat quadriceps muscle (Fig. 6,
A-C); in addition, anti-sAnk1 antibody also labeled M-lines
(Fig. 6A) (6). Cross-sections of muscle labeled with the
same panel of antibodies showed sAnk1 in a reticular pattern, consistent with its distribution in the network SR (Fig. 6D)
(6). By contrast, both titin ZIg1/2 and T-cap concentrated at the
Z-disks (Fig. 6, E and F). No labeling was
detected when primary antibodies were replaced with nonimmune rabbit or
goat IgG (Fig. 6, G and H). Thus, titin ZIg1/2
and T-cap are present at the Z-disk, whereas sAnk1 is concentrated in
the SR surrounding the Z-disk. Thus, if a complex between sAnk1 and
titin forms in skeletal myofibers, with or without T-cap/telethonin, it
would be limited to the periphery of the Z-disk.
A key question in the biology of striated muscle is how the
internal membranes of the SR and the T-tubules become precisely aligned
with the contractile apparatus. We have begun to address this question
by identifying ligands of sAnk1, a structural protein of the network SR
that, we hypothesize, helps to coordinate the alignment of the SR with
nearby M-lines and Z-disks. Because titin serves as a molecular
blueprint for the assembly of other myofibrillar elements, we
postulated that it might also provide a site for anchoring the SR
at the level of the Z-disk. We found that the hydrophilic sequence of
sAnk1, which extends from the SR membrane into the sarcoplasm (6),
specifically and directly interacted with the two most N-terminal Ig
domains of the giant myofibrillar protein titin. These two domains,
ZIg1 and ZIg2, are present in all titin muscle isoforms identified to
date and are localized at the periphery of the Z-disk lattice (47).
These domains of titin are therefore appropriately positioned to anchor
sAnk1 in the network SR to the Z-disk.
sAnk1 carries a C-terminal hydrophilic sequence that differs
significantly from the C-terminal portion of the large canonical form
of ankyrin-1. It is considerably shortened (~14 versus
~55 kDa) and contains a unique peptide sequence (amino acids 29-73); the remaining 82 residues (amino acids 74-155) are shared by
both small and large splice forms of ankyrin-1, followed by a common translation stop codon (24, 25). The results of our yeast two-hybrid
experiments suggest that a peptide 29 amino acids long (residues
61-89) contains the minimal sequence in sAnk1 with binding activity
for titin ZIg1/2. Thus, the titin-binding site on sAnk1 may be
comprised of residues unique to sAnk1 (i.e. amino acids 61-73) and amino acids shared by both small and large forms of Ank1
(i.e. amino acids 74-89). It is of course possible that the binding of sAnk1 to titin ZIg1/2 requires only some of these 29 residues. Future studies will delineate more precisely whether amino
acids that are unique to sAnk1 or shared with larger forms of Ank1 are
needed to form the titin-binding site on sAnk1.
Both the ZIg1 and ZIg2 domains of titin were required for binding to
sAnk1. This is not surprising, as the binding of many titin ligands,
such as myomesin, M-protein, myosin-binding protein C, obscurin, and
T-cap/telethonin, requires the presence of pairs of titin Ig domains
(37, 38, 41, 47, 50, 54-56). Indeed, the Ig domains of the defining
members of the Ig superfamily, the immunoglobulins, act in tandem to
form the binding sites of antibodies (57). The requirement of both ZIg1
and ZIg2 implies that the binding site of titin for sAnk1 includes
residues from both Ig domains. Preliminary observations from our
laboratory indicated that sAnk1 may homodimerize or multimerize
in vivo. Thus, a sAnk1 dimer or multimer may be the active
ligand for the ZIg1/2 region of titin. Interestingly, ZIg1 and ZIg2
share a highly conserved peptide (i.e.
SGXYS The two N-terminal Ig domains of titin were previously shown to contain
the binding site of a Z-disk protein referred to as T-cap or telethonin
(47, 50). Similar to sAnk1, the binding of T-cap to titin requires the
presence of both ZIg1 and ZIg2. Titin ZIg1/2 can bind simultaneously to
both sAnk1 and T-cap, indicating that these proteins can form a
three-way complex.
The subcellular location of these proteins places limits on where this
complex could form. T-cap and titin ZIg1/2 co-localize at the edge of
the Z-disk lattice, where their binding is believed to anchor the
N-terminal portion of titin to the Z-disk (47). By contrast, sAnk1 is
limited to the SR at the periphery of the Z-disk, where its C-terminal
hydrophilic sequence extends from the SR membrane (6).2
sAnk1 is therefore likely to interact with titin and possibly form a
three-way complex with T-cap only at the periphery of the Z-disk (Fig.
7). As postulated, this interaction would
link the network SR, where sAnk1 is concentrated, to the Z-disk.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin
within the Z-disk (47-49). The two most N-terminal Ig domains of
titin, which are constitutively expressed in all titin isoforms and
reside in the periphery of the Z-disk, bind a recently identified,
19-kDa protein of striated muscle, referred to as T-cap or telethonin
(47, 50).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin, primer G (5'-ACGTGAATTCAAGGAAACTAGGAAAACA-3', sense)
was used with primer H (5'-ACGTCTCGAGGACAGTCACATTTTTTAA-3', antisense). For ZIg4/5, immediately following the Z-repeat region, primers I (5'-ACGTGAATTCATAGAAGGTGAATCTGTC-3', sense) and
J (5'-ACGTCTCGAGTCCATGCTTATTGCGAAC-3', (antisense) were used. For
generation of MIg1/2, the first two Ig domains in the M-line region of
titin, primer K (5'-ACGTGAATTCGGTGAAAATGTCCGGTT-3', sense) was used
with primer L (5'-ACGTCTCGAGCCCAGCTGTGTTAGT-3', antisense). For MIg5/6,
two additional Ig domains in the M-line region of titin, primer M
(5'-ACGTGAATTCCTGACCTGTGTGGTTGAA-3', sense) was utilized
in combination with primer N (5'-ACGTCTCGAGTCCAGCTGAATTTTTTAC-3', antisense). All sense primers carried an EcoRI recognition
sequence, whereas all antisense primers contained an XhoI
site for insertion into the yeast two-hybrid pB42AD prey vector
(Clontech, Palo Alto, CA) and the pGEX4T-1 vector
(Amersham Biosciences) for production of glutathione
S-transferase (GST) fusion proteins. The titin ZIg1/2
fragment was also introduced into the pMAL-c2X vector at EcoRI/SalI sites (New England Biolabs Inc.,
Beverly, MA) (XhoI and SalI sites have compatible ends).
-D-thiogalactopyranoside for 3 h and
purified by affinity chromatography on glutathione-agarose (for GST
fusion proteins) (Amersham Biosciences) or amylose resin (for MBP
fusion proteins) (New England Biolabs, Inc.) columns following the
manufacturers' instructions.
-Galactosidase Assays--
The
Matchmaker LexA two-hybrid system (Clontech) was
used as recommended by the manufacturer. The pB42AD prey vector and the pLexA bait vector were used to express titin (i.e. ZIg1/2,
ZIg1/2-A, ZIg1/2-B, Zr, ZIg3, ZIg4/5, MIg1/2, and MIg5/6) and sAnk1
(i.e. sAnk1-(29-155), sAnk1-A, sAnk1-B, and sAnk1-C) hybrid
peptides, respectively, as described above. Saccharomyces
cerevisiae strain EGY48 was sequentially transformed with reporter
p8op-lacZ, bait, and prey plasmids. True transformants were selected by
plating on induction medium (i.e. synthetic dropout
Gal/Raf lacking Ura, His, Trp, and Leu) in the presence of 80 mg/liter
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal).
-galactosidase assays were performed as described in the
Clontech Yeast Protocols Handbook using
chlorophenol red
-D-galactopyranoside as substrate. For
each interaction tested, four independent colonies were assayed, and
each experiment was repeated twice. Results represent average values.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase assays (Fig. 1C) indicated that
sAnk1-(29-155) specifically interacted with the two most N-terminal Ig domains of titin, ZIg1/2 (amino acids 1-200), which reside at the edge of the Z-disk (28, 47). No specific association between sAnk1-(29-155) and the remaining ~60-kDa portion of Z-disk titin could be detected (Fig. 1C).
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Fig. 1.
Yeast two-hybrid analysis identifies titin as
a cytoplasmic ligand for sAnk1. A, sAnk1 consists of an
hydrophobic N-terminal sequence that anchors the molecule to the SR
membrane, followed by a hydrophilic sequence that is exposed on the
cytoplasmic face of the membrane. We introduced the C-terminal
hydrophilic "tail" of sAnk1 (amino acids 29-155) into the pLexA
bait vector of the Matchmaker yeast two-hybrid system. B,
consecutive PCR fragments spanning the entire length of the Z-band
region of titin (i.e. ZIg1/2, ZIg3, Zr, and ZIg4/5) or parts
of the M-line region of titin (e.g. MIg1/2 and MIg5/6) were
inserted into the pB42AD prey vector. C, yeast two-hybrid
analysis followed by liquid -galactosidase (
-gal)
assay indicated that sAnk1 specifically interacted with the two most
N-terminal Ig domains of titin, ZIg1/2. Other regions of titin from the
N-terminal region, associated with Z-disks, or the C-terminal region,
proximal to M-lines, failed to interact with sAnk1.
-galactosidase assays with different combinations of
truncated sAnk1 and titin ZIg1/2 constructs indicated that sAnk1-A
(amino acids 29-89) and sAnk1-C (amino acids 61-130) elicited similar
results, suggesting that the amino acids shared by the two subfragments
(i.e. amino acids 61-89) contain the binding site for titin
ZIg1/2 (Fig. 2A). Furthermore, both ZIg1 and ZIg2 were
required for titin to bind to sAnk1 because sAnk1 failed to interact
with the individual Ig domains (Fig. 2B).
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Fig. 2.
Identification of the binding domains of
sAnk1 and titin. To determine the regions of sAnk1 and titin
ZIg1/2 required for their association, we generated a series of
deletion constructs and introduced them into the bait and prey vectors,
respectively. A, yeast two-hybrid analysis followed by
-galactosidase (
-gal) assay showed that amino acid
residues 61-89 of sAnk1 mediate the binding of sAnk1 to titin.
B, both N-terminal titin Ig domains are required for binding
to sAnk1.
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Fig. 3.
Binding of native sAnk1 from skeletal muscle
homogenates to titin ZIg1/2. A, GST fusion proteins
containing portions of the Z-disk and M-line regions of titin
(i.e. GST-ZIg1/2, ~47 kDa; GST-ZIg3, ~65 kDa; GST-Zr,
~64 kDa; GST-ZIg4/5, ~60 kDa; MIg1/2, ~44 kDa; and MIg5/6, ~47
kDa) and control GST protein (25 kDa) were analyzed by SDS-PAGE and
visualized by staining with Coomassie Blue. B, equivalent
amounts of these recombinant proteins bound to glutathione matrices
were incubated with homogenates of adult rat quadriceps skeletal
muscle. Binding of native sAnk1 to the titin fragments was examined by
immunoblot analysis with anti-sAnk1 antibodies. Only GST-ZIg1/2
specifically retained native sAnk1.
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Fig. 4.
sAnk1 binds to titin ZIg1/2 in a blot overlay
assay. A, bacterially expressed GST-ZIg1/2 (~47 kDa)
and MBP-sAnk1-(29-155) (~56 kDa) were purified by affinity
chromatography on glutathione-agarose and amylose resin columns,
respectively; fractionated on SDS-PAGE; and stained with Coomassie
Blue. B, equivalent amounts of control GST protein and
GST-ZIg1/2 were subjected to SDS-PAGE, electrophoretically transferred
to nitrocellulose membranes, and incubated with recombinant
MBP-sAnk1-(29-155). Bound MBP-sAnk1-(29-155) was detected by Western
blotting with antibodies (ab) to sAnk1. Binding of
MBP-sAnk1-(29-155) to GST-ZIg1/2 bound to nitrocellulose was inhibited
by soluble GST-ZIg1/2 (5 and 10 µg).
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Fig. 5.
Titin ZIg1/2 binds simultaneously to sAnk1
and T-cap in vitro. A, recombinant
GST-T-cap-(1-140) (~40 kDa), GST-sAnk1-(29-155) (~39 kDa),
MBP-T-cap-(1-140) (~57 kDa), MBP-sAnk1-(29-155) (~56 kDa), and
MBP-ZIg1/2 (~64 kDa) fusion proteins were generated and analyzed by
SDS-PAGE, followed by staining with Coomassie Blue. B,
GST-sAnk1-(29-155) and control GST protein attached to glutathione
matrices were incubated with MBP-ZIg1/2 in the presence or absence of
MBP-T-cap-(1-140). Bound MBP-ZIg1/2 was assayed by SDS-PAGE and
immunoblotting with anti-titin ZIg1/2 antibody (ab).
GST-sAnk1-(29-155), but not GST, retained affinity-purified MBP-ZIg1/2
in the absence or presence of MBP-T-cap-(1-140). In a similar
experiment, GST-T-cap-(1-140), but not GST protein, attached to
glutathione-Sepharose retained MBP-ZIg1/2 in the absence or presence of
MBP-sAnk1-(29-155). C, the procedure described in
B for GST and GST-sAnk1-(29-155) was followed, except that
the matrix-bound complex was analyzed for bound MBP-T-cap-(1-140). The
complex formed by binding of GST-sAnk1-(29-155) to MBP-ZIg1/2
bound MBP-T-cap-(1-140) as shown by Western blotting with anti-T-cap
antibodies. D, the procedure described in B for
GST and GST-T-cap-(1-140) was followed, except that the matrix-bound
complex was analyzed for bound MBP-sAnk1-(29-155). The complex formed
by binding of GST-T-cap to MBP-ZIg1/2 bound recombinant
MBP-sAnk1-(29-155) as shown by immunoblot analysis with specific
antibodies to sAnk1.
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Fig. 6.
Intracellular localization of sAnk1, titin
ZIg1/2, and T-cap in adult skeletal muscle. Frozen longitudinal
sections of the quadriceps muscle of adult rats were labeled with
antibodies to sAnk1 (A), titin ZIg1/2 (B), and
T-cap (C). sAnk1 is in register with Z-disks
(arrow) and M-lines (arrowhead), whereas titin
ZIg1/2 and T-cap are present only at the level of Z-disks
(arrows). In cross-sections of the same muscle labeled with
the same panel of antibodies, sAnk1 is present in a reticulum
surrounding Z-disks and M-lines (D), whereas titin ZIg1/2
(E) and T-cap (F) reside in the plaque-like
structures that are the Z-disks. The patterns observed in
A-F were absent when primary antibodies were replaced with
nonimmune rabbit (G) or goat (H) IgG, suggesting
that the labeling is specific.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
XATN, where X is a
nonconserved amino acid and
is a nonpolar hydrophobic residue) that
could serve as the binding site of a potential sAnk1 dimer. Although further experimentation will be required to address this issue, the
results of our in vitro binding studies suggest that
dimerization of sAnk1 may not be required for it to bind to titin
ZIg1/2.
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Fig. 7.
Model of a complex containing sAnk1, titin
ZIg1/2, and T-cap at the periphery of the Z-disk. sAnk1 is
an integral membrane protein of the network SR. It is anchored to the
membrane by its hydrophobic N-terminal sequence, leaving its C-terminal
hydrophilic tail extending into the sarcoplasm at the level of Z-disks
and M-lines. Titin is a giant myofibrillar protein that spans the
half-sarcomere, with its N terminus near the Z-disk and its C terminus
embedded in the M-line. Residues 1-200 of titin contain two Ig domains
(ZIg1 and ZIg2) that reside at the periphery of the Z-disk and that are
constitutively expressed in all known isoforms of titin. Both ZIg1 and
ZIg2 are required for a specific, direct, and presumably physiologic
association with the cytoplasmic sequence of sAnk1 as well as with the
Z-disk protein T-cap. sAnk1 does not compete with T-cap for binding to
titin ZIg1/2, suggesting that a complex of these three proteins can
form in situ. However, their relative locations suggest that
a three-way complex can form only at the periphery of the Z-disk. The
binding of sAnk1 to titin ZIg1/2, whether the latter is also bound to
T-cap or not, implies a role for these proteins in coordinating the
organization of the network SR with that of nearby contractile
structures.
T-cap was recently shown to bind to MinK, the -subunit of the
potassium channel of the transverse tubular membranes (58). This
suggests that, just as sAnk1 links titin at the periphery of the Z-disk
to the SR, T-cap may link the same population of titin molecules to the
T-tubules. These interactions can occur in mammalian cardiac muscle and
in avian and amphibian striated muscle, where both the SR and T-tubules
are located around the Z-disk. In mammalian skeletal muscle, however,
this function is likely to be limited to sAnk1, as only the SR is
concentrated around the Z-disk. The T-tubules in skeletal muscle are
present at the junction of the A- and I-bands and so are unlikely to be anchored via T-cap to titin at the Z-disk. Thus, through its ability to
bind simultaneously to sAnk1 and T-cap, titin may simultaneously serve
as a scaffold for assembling not only the contractile apparatus, but
also the network SR and, in many (but not all) striated muscles, the
T-tubules.
Titin is the second myofibrillar protein that we have identified as a major cytoplasmic ligand for sAnk1: obscurin also binds to sAnk1 (53). Obscurin is a giant (~800 kDa) sarcomeric Rho guanine nucleotide exchange factor protein with homology to titin (55). Immunolocalization studies have shown that obscurin closely surrounds the myofibrils at the Z-disks and M-lines of each sarcomere (53). Thus, it appears that, whereas titin associates with sAnk1 at the level of the Z-disk, obscurin interacts with sAnk1 around both Z-disks and M-lines.
We have previously shown that amino acids 61-130 of the C-terminal
hydrophilic sequence of sAnk1 contain the binding site for obscurin
(53). In the present study, we have shown that residues 61-89 of sAnk1
are likely to contain the binding site for titin ZIg1/2. These findings
suggest that the sAnk1 binding sites for titin and obscurin may
overlap. Future studies will examine whether titin and obscurin compete
with each other for binding to sAnk1 around the Z-disk in developing or
mature striated muscle and whether one or both of these giant
sarcomeric proteins play an important role in aligning the SR with the
contractile apparatus.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. S. Labeit and C. C. Gregorio for providing the full-length pET9D-T-cap plasmid and Dr. C. C. Gregorio for sharing anti-titin-x112 with x113 antibody. We also thank W. G. Resneck and A. O'Neill for expert assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant RO1 HL64304 (to R. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of National Institutes of Health Fellowship T32
AR07293. To whom correspondence should be addressed: Dept. of
Physiology, University of Maryland School of Medicine, 685 W. Baltimore
St., Baltimore, MD 21201. Tel.: 410-706-4410; Fax: 410-706-8341;
E-mail: akons001@umaryland.edu.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M209012200
2 N. Porter, W. Resneck, A. O'Neill, D. van Rossum, and R. J. Bloch, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: T-tubules, transverse tubules; SR, sarcoplasmic reticulum; sAnk1, small ankyrin-1; GST, glutathione S-transferase; MBP, maltose-binding protein.
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