From the Mayo Foundation, S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259
Received for publication, May 1, 2000, and in revised form, September 12, 2000
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ABSTRACT |
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Some disease-associated truncations within the
100-residue domain C-terminal of the second nucleotide-binding domain
destabilize the mature protein (Haardt, M., Benharouga, M., Lechardeur,
D., Kartner, N., and Lukacs, G. L. (1999) J. Biol.
Chem. 274, 21873-21877). We now have identified three
short oligopeptide regions in the C-terminal domain which impact cystic
fibrosis transmembrane conductance regulator (CFTR) maturation
and stability in different ways. A highly conserved hydrophobic patch
(region I) formed by residues 1413-1416 (FLVI) was found to be crucial
for the stability of the mature protein. Nascent chain stability was
severely decreased by shortening the protein by 81 amino acids (1400X).
This accelerated degradation was sensitive to proteasome inhibitors but
not influenced by brefeldin A, indicating that it occurred at the
endoplasmic reticulum. The five residues at positions 1400 to 1404 (region II) normally maintain nascent CFTR stability in a positional
rather than a sequence-specific manner. A third modulating region (III) constituted by residues 1390 to 1394 destabilizes the protein. Hence
the nascent form regains stability on further truncation back to
residues 1390 or 1380, permitting some degree of maturation and a low
level of cyclic AMP-stimulated chloride channel activity at the cell
surface. Thus while not absolutely essential, the C-terminal domain
strongly modulates the biogenesis and maturation of CFTR.
The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is a large
multidomain membrane protein that forms a tightly regulated chloride
channel in the apical membrane of many chloride secreting and
reabsorbing epithelial cells (1, 2). As an adenine nucleotide binding
cassette (ABC) protein it contains two nucleotide-binding domains (NBD1
and NBD2) and two transmembrane domains (TMD1 and TMD2), each
spanning the membrane several times. CFTR also has a large R-domain
between NBD1 and TMD2 which is not common to other ABC proteins and is
the site of regulatory phosphorylation and dephosphorylation (3-6).
Additionally there are N- and C-terminal cytoplasmic domains of less
than 100 amino acids each that precede TMD1 and follow NBD2,
respectively. The former has recently been shown to participate in
channel regulation by interacting with the R-domain (7). The C-terminal
domain, however, is apparently not essential to channel function
(8-10) but the very C terminus can tether the protein to PDZ domain
containing proteins (11, 12), possibly to localize CFTR within
regulatory complexes.
Wild-type CFTR matures very inefficiently following synthesis on
membrane-bound ribosomes (13) and many disease-associated mutations in
different domains preclude the formation of any mature protein that can
be transported to the cell surface. Hence, it is important to
understand the role of each domain of the molecule in achieving and
maintaining a mature and stable conformation. Frameshift or premature
stop mutations found in patients with cystic fibrosis that cause
truncations at several locations in the C-terminal domain were shown to
destabilize the mature CFTR protein so that its lifetime was greatly
shortened (10). We have now made systematic stepwise truncations as
well as deletions and substitutions across the entire C-terminal domain
and identified different short sequences which strongly influence not
only the stability of the mature protein but also the maturation and
stability by the nascent chain. The major determinant of the steady
state amount of mature protein is a hydrophobic "patch" formed by
residues 1413 to 1416 whereas there are strong "positional effects"
on the nascent chain of sequences closer to NBD2. Truncation back to
residue 1400 resulted in an extremely unstable nascent chain, degraded
at the endoplasmic reticulum (ER) by the proteasome. Strikingly, on
further truncation back to residues 1390 or 1380 the nascent protein
regains stability such that some mature protein is again formed that
mediates a low but detectable level of cAMP-stimulated chloride efflux.
These findings reveal that C-terminal motifs and their positioning have
a major impact on the assembly and stability of the CFTR ion channel.
Plasmid Construction--
All C-terminal truncations were
constructed by polymerase chain reaction introducing a TAG stop
codon flanked by an ApaI restriction site. The polymerase
chain reactions were performed on pBQ4.7 CFTR plasmid DNA. The
antisense primers introducing the stop codon were the following:
A1440X, 5'-TGATATCGGGCCCCTATTGCCGGAAGAGGCTCCTCTCGTT-3'; S1435X,
5'-TGATATCGGGCCCCTACCTCTCGTTCAGCAGTTTCTGGATGG-3'; L1430X, 5'-TGATATCGGGCCCCTATTTCTGGATGGAATCGTACTGCCGCAC-3'; D1425X,
5'-TGATATCGGGCCCCTAGTACTGCCGCACTTTGTTCTCTTCTATG-3'; K1420X,
5'-TGATATCGGGCCCCTAGTTCTCTTCTATGACCAAAAATTGTTGGC-3'; V1415X, 5'-TGATATCGGGCCCCTACAAAAATTGTTGGCATTCCAGCATTGC-3'; C1410X,
5'-TGATATCGGGCCCCTATTCCAGCATTGCTTCTATCCTGTGTTC-3'; E1405X,
5'-TGATATCGGGCCCCTATATCCTGTGTTCACAGAGAATTACTGTGC-3'; C1400X, 5'-TGATATCGGGCCCCTAGAGAATTACTGTGCAATCAGCAAATGC-3'; E1405X,
5'-TGATATCGGGCCCCTATATCCTGTGTTCACAGAGAATTACTGTGC-3'; C1395X,
5'-TGATATCGGGCCCCTAATCAGCAAATGCTTGTTTTAGAGTTCTTC-3'; Q1390X, 5'-TGATATCGGGCCCCTATTTTAGAGTTCTTCTAATTATTTGGTATGTTAC-3'; T1380X, 5'-TGATATCGGGCCCCTATACTGGATCCAAATGAGCACTGGGTTC-3'. The sense
primer was derived from nucleotides 3551-3579
(5'-TGAGTACATTGCAGTGGGCTGTAAACTCC-3'). The fragments generated by
polymerase chain reaction were subcloned into pBluescript
SK+. The BprPI-ApaI fragment of the
created plasmids was then used to replace the corresponding fragment of
pNUT-D1270V CFTR. This plasmid, derived from pNUT-CFTR, contains an
engineered ApaI site at the 3' end of
CFTR.2
Replacements of various amino acids with alanine and the deletion of
amino acids 1400 to 1404 ( Stable Expression of Mutant and Wild-type CFTR in BHK
Cells--
Baby hamster kidney cells grown at 37 °C in 5%
CO2 were stably transfected and analyzed as described
earlier (15).
Cell Surface Labeling of CFTR--
Cells were washed twice with
PBS containing 0.1 M CaCl2 and 1 mM
MgCl2 (PBS++) and incubated for 2 min with 10 mM sodium periodate in PBS++ in the dark. Cells were washed
twice with 0.1 M sodium acetate, pH 5.5, with 0.1 mM CaCl2 and 1 mM MgCl2
and incubated for 1 min with 1 mM biotin-LC-hydrazide
(Pierce), in the same buffer, in the dark. The labeling buffer was
aspirated and cells were incubated for 2 min in 0.1 M
Tris-HCl, pH 7.5, with 1% bovine serum albumin, 0.1 M
CaCl2, and 1 mM MgCl2 to stop the
reaction. After washing 5 times with ice-cold PBS++, cells were lysed
and biotinylated proteins were isolated with immobilized streptavidin (Pierce) as we have described recently (17).
Metabolic Pulse-Chase Labeling--
Stably transfected BHK-21
cells were starved in methionine-free medium for 30 min, labeled for 20 min with 0.1 mCi/ml [35S]methionine, and chased with
Dulbecco's modified Eagle's medium/F-12 supplemented with 1 mM methionine and 5% fetal bovine serum. Cell lysis and
immunoprecipitation were performed as described before (15). With the
exception of the 1380X CFTR mutant, all immunoprecipitations were
performed using the monoclonal antibody M3A7 (16). Since the molecule
1380X does not contain the complete M3A7 epitope, it was
immunoprecipitated with the L12B4 antibody (16). The amount of
35S-radioactivity in each band was quantified by electronic
autoradiography using a Packard Instant Imager.
Treatment with Brefeldin A and Protease
Inhibitors--
Brefeldin A (BFA), lactacystin,
N-acetyl-leucinyl-leucinyl-norleucinal (ALLN),
N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), and
4-hydroxy-5-iodo-3-nitrophenylacetyl-leucinyl-leucinyl-leucinyl-vinylsulfone (NLVS) were added to cells 90 min prior to methionine starvation to a
final concentration of 10 µg/ml (BFA) and 10 µM (NLVS)
or 50 µM (lactacystin, MG132, and ALLN) and were present
during the pulse labeling and chase period.
Immunofluorescence Microscopy--
Cells were grown on
coverslips and fixed in 100% methanol at 36Cl Influence of C-terminal Truncations on Steady State Amounts of
Immature and Mature CFTR--
Haardt et al. (10) had
previously shown that truncations at several positions within the
C-terminal domain destabilized mature CFTR. We decided to further
elucidate sites within the C-terminal domain that impact maturation and
stability of both nascent and mature CFTR. As a first step, systematic
incremental truncations terminating at the points indicated in Fig.
1A were constructed and
expressed. As shown in Fig. 1B the most C-terminal 40 residues of the CFTR sequence distinguish it entirely from other
related human ABC proteins. The final four residues of this CFTR-specific segment form a PDZ domain binding motif (11, 19, 20),
which may contribute to apical localization (21) and be involved in the
correct placement of CFTR within multimolecular regulatory complexes
(12, 22, 23). No function has yet been attributed to the remainder of
this 40 residue block (residues 1440-1476). Fig. 1C shows
that truncation at this point to form a 1440X variant had no apparent
effect on the steady state amounts of the immature, core-glycosylated
form of CFTR (lower band) or the mature form with complex
oligosaccharide chains (upper band). Apparently no major
changes in these amounts occur when truncation of as many as 61 residues occurs, i.e. to form 1420X. However, truncation of
a further five residues from this point resulted in a major reduction
in the amount of the mature band (1415X). Shortening the C-terminal
region by an additional five residues (1410X) nearly eliminated the
mature protein band. Hence a portion of the polypeptide between
residues 1410 and 1419 appears to play an important role in the
formation or maintenance of mature CFTR. The intensity of the immature
band also appeared to be significantly reduced on shortening from 1415X
to 1410X (Fig. 1C). The next three incremental shortenings
to 1405X, 1400X, and 1395X were without apparent additional effect; the
mature band was barely detectable and the immature band remained
fainter when compared with 1415X and was weakest at 1400X.
Strikingly, deeper truncation to 1390X resulted in a significant
increase in the amounts of both bands, the immature band appearing
nearly as strong as in the much longer constructs and the mature band
as strong as at 1415X. Further shortening to 1380X resulted in a still
larger amount of the mature band. To ensure that the mature form of
CFTR reach the plasma membrane surface, labeling was carried out with
the membrane-impermeable reagent biotin-LC-hydrazide (Fig.
1D). Mature forms of 1390X CFTR and 1380X CFTR were found to
be biotinylated. These observations suggest that some sequence
C-terminal of residue 1390 is actually destabilizing and emphasizes the
fact that CFTR biogenesis and maturation can occur reasonably
effectively in the complete absence of the C-terminal domain beyond
NBD2. However, certain short regions within the domain have strong
modulating effects, both positive and negative. At least three
different short segments of the C-terminal domain have significant
impact on the steady state amounts of immature and mature CFTR observed.
Immunofluorescence Localization of C-terminal Truncated CFTR
Variants--
The influence of these truncations on the intracellular
localization of CFTR in stably transfected BHK-21 cells was evaluated by immunofluorescence microscopy. Simultaneous staining with an antibody to the plasma membrane Ca2+-ATPase showed that
wild-type CFTR is detectable as a uniform staining over the entire cell
surface, some of which is punctate in nature (Fig.
2A). Clearly visible beneath
the surface staining was a more intense perinuclear pattern
corresponding well with ER localization in these cells. Overall this
distribution was similar to that observed with CFTR expression in other
nonpolar mammalian cells and the same as that observed in BHK cells
expressing a CFTR-green fluorescent protein fusion (15). Truncation to remove the last 41 C-terminal amino acids (1440X) did not change the
wild-type picture (Fig. 2B). However, shortening by 20 more residues to 1420X resulted in some decrease in cell surface staining while perinuclear staining remained intense. With the 1400X truncation only a very circumscribed perinuclear pattern was seen, consistent with
the presence of primarily the core-glycosylated immature band seen in
immunoblots (Fig. 1) and immunoprecipitates (Fig. 3). Shortening by a further 10 or 20 residues to 1390 or 1380 caused the reappearance of weak staining over
the cell surface. Hence there was good correspondence between the
relative amount of the mature CFTR band detected in Western blots of
whole cell lysates (Fig. 1C) and cell surface staining by
immunofluorescence among the different truncations (Fig. 2).
Influence of C-terminal Truncations on the Turnover of Immature and
Mature Forms of CFTR--
Pulse-chase experiments were performed to
determine the kinetic effects of each of these truncations on nascent
chain maturation or degradation and on the lifetime of the mature
molecule once it was formed (Fig. 3). As has been consistently observed
in a variety of different mammalian cell types, wild-type CFTR matures inefficiently with 40% or less of the pulse-labeled immature band converted to the mature band during the chase. This maximal conversion occurs in ~2 h after which little or no immature band is detectable and the amount of radioactively labeled mature band remains nearly constant until 4 h since it has a half-time of ~16 h in these cells (13, 24). Other observations showed that the rate of disappearance of the immature band is slowed by proteasome inhibitors without augmenting the amount that is converted to the mature product
(25, 26) suggesting that a large proportion (in this case ~60%) of
nascent chain was degraded by the proteasome. Truncations to produce
1440X, 1435X, 1430X, and 1425X did not drastically change this
precursor-product relationship of nascent and mature CFTR. Notably the
maximal proportion of the pulse-labeled nascent 1420X that matured was
closer to 30% than 40% and the mature band decayed somewhat more
rapidly than wild-type. As was already apparent from the Western blots
(Fig. 1) truncation to 1415X had a more major impact. In the
pulse-chase experiment the immature band disappeared somewhat more
slowly, less mature band appeared and it then turned over more rapidly.
The rates of nascent chain disappearance and mature form appearance and
disappearance were similar to that of 1410X and 1405X, although the
maximum proportion of mature protein formed decreased progressively
with shortening. By 1400X this proportion was minimal (~7% at 1 h of chase). Strikingly, despite the fact that conversion to the mature
form was barely detectable, the immature 1400X band disappeared
extremely rapidly, i.e. shortening from 1405X to 1400X
greatly accelerated the rate of disappearance of the nascent chain.
Unexpectedly, removal of a further 5 residues to produce 1395X seemed
to cause a reversion to a situation more similar to 1405X than 1400X. A
more pronounced restabilization and maturation was exhibited by 1390X.
In fact the curve showing the rates of disappearance of the immature
band were again quite similar to much longer variants including the
wild-type as if residues C-terminal of 1390 destabilized the nascent
chain. Formation and maintenance of mature 1390X, however, remained
considerably depressed. Further shortening to 1380X, essentially to the
C-terminal end of NBD2, had little additional effect.
Overall these kinetic data confirm and extend the detection by the
immunoblots of three different short segments, within the N-terminal
portion of the C-terminal 100 residue domain of CFTR, which have major
influences on the stability of the nascent and mature forms of the
molecule. These segments lay within a 40-residue stretch immediately
C-terminal of NBD2; the most C-terminal 60 residues have little
influence on the turnover of immature and mature CFTR and presumably
play other roles.
The three short segments delineated by the deletions are highlighted in
Fig. 4A and are seen to be
reasonably well conserved in CFTRs from different species. Numbering
from the C-terminal end, these segments include region I, which when
removed greatly reduced mature CFTR, consisting of amino acids 1413 to
1417, all of which except 1417E are hydrophobic, region II (1400-1404)
which seems necessary for nascent chain stability, and region III
(1390-1394) which has the opposite effect, i.e.
destabilizes the nascent molecule.
The "Hydrophobic Patch" of Residues 1413-1416 in Region I Is
Essential for the Formation and Maintenance of Mature CFTR--
Haardt
et al. (10) reported that naturally occurring C-terminal
truncations increased the rate of turnover of the mature form. We
observed that C-terminal truncation of 66 amino acids or more decreased
the amount of mature protein drastically. Our truncations in steps of
five residues suggested the involvement of region I. When all five
residues from 1413 to 1417 (FLVIE) were replaced by alanine, the effect
was essentially the same as truncation after position 1410, i.e. no mature CFTR band was detected by Western blotting
(Fig. 4B). To determine the crucial residues within region
I, alanine substitutions were also made in pairs and individually. Each
of the four contiguous hydrophobic residues appeared to contribute and
substitution of the Phe1413-Leu1414 pair by
alanines was nearly as detrimental as removing all four. Hence a short
hydrophobic patch of at least two residues seems to be required for the
appearance of the mature protein. Substitution of the glutamic acid at
position 1417 was entirely without effect as was replacement of the
nonconserved glutamate at 1418 and substitution of the two glutamines
flanking this patch on the N-terminal side (Fig. 4A). In
pulse-chase experiments just traces of the large band with complex
oligosaccharides could be detected (less than 5%) which turned over
rapidly (Fig. 4D). The nascent chain, however, persisted
with a half-life longer than the wild-type protein. The absence of
mature CFTR on substitution of this patch of hydrophobic residues was
also observed by immunofluorescence microscopy where no cell surface
staining could be detected (Fig. 4C).
Residues 1400-1404 (CEHRI) in Region II Contribute to Nascent CFTR
Stability in a Positional Rather than a Sequence-specific
Manner--
The pulse-chase experiments in Fig. 3 had shown that
nascent CFTR became extremely unstable (short-lived) on truncating at residue 1400. Further evidence that this truncation influences nascent
CFTR turnover at the ER came from the fact that its rapid disappearance
was not significantly influenced by treatment of cells with BFA (Fig.
5A) which is known to inhibit
the trafficking of CFTR out of the ER without blocking conformational
maturation (27, 28). The ubiquitin-proteasome pathway was shown to be responsible for the degradation of immature wild-type CFTR (25, 26). To
study the role of the 26 S proteasome in the degradation of 1400X CFTR,
we investigated the turnover of the truncated protein in the presence
of various proteasome inhibitors in BFA-treated cells (Fig.
5B). The highly specific proteasome inhibitor lactacystin strongly stabilized the truncated protein. A significant delay in the
turnover of 1400X CFTR was also achieved by the reversible peptide
aldehyde inhibitors ALLN or MG132 and by the peptide vinyl sulfone
NLVS, which recently has been demonstrated to be a useful inhibitor of
the proteasome (29). In direct contrast to the influence of truncation
after residue 1400, replacement of amino acids 1400 to 1404 with
alanines allowed the nascent chain to be nearly as stable and mature
nearly as effectively as wild-type (Fig. 5C). Thus these
five residues must play an essential positional role rather than
providing a precise sequence. This interpretation was supported by
substituting these residues with alanines pairwise which yielded steady
state amounts of immature and mature protein similar to wild-type and
is also strengthened by the observation that deletion of this region
( Residues 1390-1394 (QAFAD) in Region III Destabilize Nascent
CFTR--
The continued incremental truncation of CFTR beyond residue
1395 had the unexpected effect of partially restoring the stability and
maturation of nascent CFTR (Figs. 1-3), i.e. 1390X was more like wild-type than 1395X. Alanine substitutions en bloc were again
used to assess the nature of the destabilizing effect of the residues
between these points. The alanines seemed to serve as effectively as
the native sequence to support stability and maturation (Fig.
6). Hence it appears that termination of
the polypeptide with the native 1390-1394 sequence has a very negative effect which is not manifest when it is followed by more of the normal
sequence. The fact that reasonable maturation and stability can be
achieved in the entire absence of the last 91 or 101 residues of CFTR
probably means that parts of this domain play different essential roles
not fundamentally involved with determining the lifetime of either form
of the protein.
Chloride Channel Activity of C-terminal Mutants--
As mentioned,
earlier work had already shown that CFTR retained some chloride channel
activity after truncations of substantial portions of the C-terminal
domain (8-10). However, we wished to determine whether those mutations
having greatest impact on processing and stability had effects on
channel function other than what might be expected from the steady
state amounts of protein present in each case. Hence
36Cl The function of the 100-amino acid domain of CFTR, C-terminal of
the second nucleotide-binding domain, has not been defined. Notably it
extends ~40 residues beyond the C terminus of other human ABC
proteins, even those of the same subfamily (Fig. 1B). The
final four residues, DTRL bind to PDZ domain containing proteins (11,
12). This interaction has been proposed to position CFTR within
multimolecular regulatory complexes (22, 23), possibly contributing to
its control by protein kinase A (30-32) and enabling it to influence
or respond to other proteins involved in epithelial salt transport such
as epithelial Na+ conductance, the epithelial sodium
channel (33, 34). Removing or substituting the terminal PDZ
domain-binding amino acids has also been reported to prevent apical
localization in epithelial cells (21, 35), although another ABC
protein, P-glycoprotein, localizes to apical membranes (36, 37) without
having a PDZ domain binding terminus. The studies that we have reported
here indicate that neither the final four residues nor the remainder of
the 40-residue extension play an important role in the biosynthetic processing and stability of wild-type CFTR.
Furthermore, the next 20 residues in the N-terminal direction similarly
have little influence despite the fact that they include two
trafficking signals. The tyrosine residue at position 1424 has been
reported to be involved in CFTR endocytosis (38, 39) and the dileucine
pair at positions 1430 and 1431 is comparable to those of the
structurally related SUR1 protein and has been described as essential
to its maturation and movement from the ER to the cell surface (40). In
addition to the lack of influence of truncation at residue 1420, alanine substitution of Tyr1424 and
Leu1430-Leu1431 also did not alter the steady
state amount of nascent or mature CFTR (not shown). Hence the final 61 residues of CFTR apparently are not crucially involved in its
biosynthetic assembly and stability.
In contrast, within the next 30 residues in the N-terminal direction
there are at least three short stretches that strongly impact the
turnover of both immature and mature CFTR. Hydrophobic residues at
positions 1413-1416 (region I) appear to be essential for the
formation and maintenance of the mature protein. Substitution of as few
as two of these residues prevents the appearance of mature CFTR. It is
notable that hydrophobic residues are conserved at these four positions
in all members of the CFTR/multidrug resistance-associated protein
subfamily of ABC membrane proteins. Within the hydrophobic patch, the
FL pair has the most striking impact. There are examples of pairs of
hydrophobic residues mediating specific interactions of some membrane
proteins with the secretory pathway machinery. The retrieval from the
Golgi of type I proteins, especially those of the p24 family is
dependent on binding to COPI mediated by a diphenylalanine pair in
their cytoplasmic tails (41). Although the lipophilic residues in
region I of CFTR might be needed for a crucial hydrophobic interaction,
we have not yet determined if this is intra- or inter-molecular.
Truncation to residue 1400 or deletion of amino acids 1400-1404 caused
lability of nascent CFTR but alanine substitutions of these residues
did not. Hence this region must provide correct positioning of other
parts of the domain rather than specific sequence information. In the
absence of this positional effect, the nascent chain becomes more
susceptible to degradation. The third short region comprised of
residues 1390 to 1394 apparently also imparts a sequence-independent
effect but in the opposite direction, i.e. termination of
the molecule with these five residues causes it to be extremely
unstable. When they are then removed, there is a degree of restoration
of stability. A larger amount of nascent 1390X protein is present at
steady state; it is degraded less rapidly than 1395X or 1400X and
matures more effectively than they do.
Haardt et al. (10) considered disease-associated truncations
at various sites in the C-terminal domain to be a novel class of
mutations in which the defect is entirely due to accelerated degradation of the mature molecule. Indeed the premature stop, A1411X
does remove region I and causes rapid turnover of mature CFTR. However,
the frameshift 4326de1TC resulting in truncation at residue 1398 and
the premature stop L1399X also remove region II and hence also
compromise the stability and ability to mature of the nascent chain. We
observed a stabilizing effect of truncations at residues 1390 or
deeper. But even if a small amount of mature protein could be detected,
the cAMP-stimulated chloride channel activity was very low and delayed
when compared with wild-type responses. The small amount of mature
protein may still be inadequate to allow sufficient chloride transport
across the plasma membrane and this might explain why patients carrying
frameshift 4279 insA, causing termination after residue 1384, have
severe disease. It appears that all C-terminal truncation mutations do
not fit into a single class. Our findings indicate that the effects are
very much dependent on the precise location of the truncation. Another truncation detected in a family where the sweat gland may be the sole
tissue effected, S1455X (42), would not influence the turnover of
either form of CFTR. It does, however, remove the terminal PDZ domain
binding motif and the loss of interactions mediated through it may be
responsible for the phenotype (21).
In summary, these experiments have shown that although the C-terminal
domain is not absolutely essential for the biosynthetic maturation of
CFTR, several short sequences within the region closest to NBD2 have
strong modulating effects. A major determinant of the lifetime of
mature CFTR has been localized to a highly conserved hydrophobic patch
65 residues before the end of the molecule. The precise localization of
this determinant now will allow pursuit of its mechanistic role in the
assembly and stability of the CFTR channel.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1400-1404) were constructed using the
QuickChange Site-directed Mutagenesis kit from Stratagene. A modified
pBQ4.7 (M1475V CFTR, with CFTR coding sequence identical to the one in
pBQ6.2 (14)) was utilized as a template for the polymerase chain
reaction mutagenesis. The following oligonucleotides were used to
introduce the indicated mutations into wild-type CFTR cDNA.
F1413A/L1414A/V1415A/I1416A/E1417A,
5'-GCTGGAATGCCAACAAGCTGCGGCCGCAGCAGAGAACAAAGTGCGG-3' and
5'-CCGCACTTTGTTCTCTGCTGCGGCCGCAGCTTGTTGGCATTCCAGC-3';
F1413A/L1414A/V1415A/I1416A, 5'-GCTGGAATGCCAACAAGCTGCGGCCGCAGAAGAGAACAAAGTGCGG-3' and
5'-CCGCACTTTGTTCTCTTCTGCGGCCGCAGCTTGTTGGCATTCCAGC-3'; Q1411A/Q1412A,
5'-GGATAGAAGCAATGCTGGAATGCGCAGCATTTTTGGTCATAGAAG-3 and
5'-CTTCTATGACCAAAAATGCTGCGCATTCCAGCATTGCTTCTATCC-3; F1413A/L1414A, 5'-GCTGGAATGCCAACAAGCTGCGGTCATAGAAGAGAACAAAGTGCG-3' and
5'-CGCACTTTGTTCTCTTCTATGACCGCAGCTTGTTGGCATTCCAGC-3'; L1414A/V1415A,
5'-GCTGGAATGCCAACAATTTGCGGCCATAGAAGAGAACAAAGTGCGG-3' and
5'-CCGCACTTTGTTCTCTTCTATGGCCGCAAATTGTTGGCATTCCAGC-3'; V1415A/I1416A, 5'-GGAATGCCAACAATTTTTGGCCGCAGAAGAGAACAAAGTGCGGCAG-3' and
5'-CTGCCGCACTTTGTTCTCTTCTGCGGCCAAAAATTGTTGGCATTCC-3'; E1417A/E1418A,
5'-GCCAACAATTTTTGGTCATAGCAGCGAACAAAGTGCGGCAGTACG-3' and
5'-CGTACTGCCGCACTTTGTTCGCTGCTATGACCAAAAATTGTTGGC-3'; F1413A, 5'-GCAATGCTGGAATGCCAACAAGCTTTGGTCATAGAAGAGAAC-3' and
5'-GTTCTCTTCTATGACCAAAGCTTGTTGGCATTCCAGCATTGC-3'; L1414A,
5'-GCTGGAATGCCAACAATTTGCGGTCATAGAAGAGAACAAAGTGCG-3' and 5'-CGCACTTTGTTCTCTTCTATGACCGCAAATTGTTGGCATTCCAGC-3'; V1415A,
5'-GGAATGCCAACAATTTTTGGCCATAGAAGAGAACAAAGTGCGGCAG-3' and
5'-CTGCCGCACTTTGTTCTCTTCTATGGCCAAAAATTGTTGGCATTCC-3'; I1416A, 5'-GGAATGCCAACAATTTTTGGTCGCAGAAGAGAACAAAGTGCGGCAG-3' and
5'-CTGCCGCACTTTGTTCTCTTCTGCGACCAAAAATTGTTGGCATTCC-3'; E1417A,
5'-GCCAACAATTTTTGGTCATAGCAGAGAACAAAGTGCGGCAGTACG-3' and 5'-CGTACTGCCGCACTTTGTTCTCTGCTATGACCAAAAATTGTTGGC-3';
C1400A/E1401A/H1402A/R1403A/I1404A, 5'-GCACAGTAATTCTCGCTGCAGCCGCGGCAGAAGCAATGCTGGAATGCC-3' and
5'-GGCATTCCAGCATTGCTTCTGCCGCGGCTGCAGCGAGAATTACTGTGC-3';
1400-1404:
5'-GCATTTGCTGATTGCACAGTAATTCTCGAAGCAATGCTGGAATGCC-3' and
5'-GGCATTCCAGCATTGCTTCGAGAATTACTGTGCAATCAGCAAATGC-3'; C1400A/E1401A, 5'-GATTGCACAGTAATTCTCGCTGCACACAGGATAGAAGCAATGC-3' and
5'-GCATTGCTTCTATCCTGTGTGCAGCGAGAATTACTGTGCAATC-3'; H1402A/R1403A,
5'-CAGTAATTCTCTGTGAAGCCGCGATAGAAGCAATGCTGGAATGCC-3' and
5'-GGCATTCCAGCATTGCTTCTATCGCGGCTTCACAGAGAATTAC TG-3'; I1404A/E1405A, 5'-CTCTGTGAACACAGGGCAGCAGCAATGCTGGAATGCCAAC-3' and
5'-GTTGGCATTCCAGCATTGCTGCTGCCCTGTGTTCACAGAG-3', Q1390A/A1391A/F1392A/A1393A/D1394A,
5'-GAAGAACTCTAAAAGCAGCAGCTGCTGCTTGCACAGTAATTCTC-3' and
5'-GAGAATTACTGTGCAAGCAGCAGCTGCTGCTTTTAGAGTTCTTC-3'. The
BprPI-ApaI fragment of pBQ 4.7 M1475VCFTR
and the derived mutated plasmids were used to replace the corresponding
fragment of pNUT-D1270V CFTR. The sequences of all inserted fragments
were confirmed by DNA sequencing.
20 °C for 10 min. CFTR
was detected by indirect immunofluorescence as described previously
(17) using the mouse monoclonal antibody M3A7 (16) or BJ570 which
recognizes the R-domain3 and
visualized with Alexa Fluor 488 goat anti-mouse IgG conjugate (dilution
1:250, Molecular Probes). The plasma membrane Ca2+-ATPase
was detected by the mouse monoclonal antibody 5F10 (Affinity Bioreagents, Inc.) and visualized with Alexa Fluor 594 goat anti-mouse IgG conjugate (dilution 1:250, Molecular Probes). To visualize CFTR and
the plasma membrane ATPase simultaneously, the mouse monoclonal
anti-CFTR antibody BJ570 was biotinylated with NHS-biotin (Pierce) as
described by Harlow and Lane (18) and detected with streptavidin Alexa
Fluor 488 conjugate (dilution 1:1500, Molecular Probes).
Efflux Assay--
The chloride
efflux assay was performed exactly as we have described recently (17).
Cells grown in 6-well culture dishes were loaded with loading buffer
containing 0.5 µCi of Na36Cl (Amersham Pharmacia
Biotech), washed with efflux buffer, and stimulated with
forskolin-containing buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
C-terminal domain. A, schematic
two-dimensional depiction of CFTR domain structure. CFTR is composed of
two parts, each containing six membrane-spanning domains and a NBD,
which interacts with ATP. The two parts are linked by a regulatory
(R) domain. The C terminus (CT) extends beyond
the second NBD by about 100 amino acids and is represented oversized to
indicate the amino acid positions where stop codons were introduced.
B, alignment of sequence of CFTR C-terminal domain with
other members of the same subfamily of human ABC proteins starting with
the NBD2 Walker B aspartic acid. C, influence of stepwise
C-terminal truncations on the steady state amounts of core-glycosylated
nascent CFTR (lower band) and the mature protein with
complex oligosaccharide chains (upper band). CFTR cDNA
constructs with stop codons at the positions indicated by X
were employed for stable expression in BHK-21 cells as described
previously (15). 10 µg of total cell lysates were electrophoresed and
Western blots probed with the monoclonal antibody, L12B4 (16).
D, cell surface labeling of mature CFTR. The surface sugars
of stable transfected BHK-21 cells were oxidized and covalently linked
to biotin-LC-hydrazide (Pierce). Biotinylated proteins were purified
with immobilized streptavidin (Pierce) and Western blotting was
performed with L12B4 (16).
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Fig. 2.
Immunofluorescence localization of wild-type
and C-terminal truncations of CFTR. Stably transfected
BHK-21 cells grown on glass slides were fixed in 100% methanol,
permeabilized in 0.1% saponin, and visualized by indirect
immunofluorescence as described previously (17). A, CFTR
localizes to plasma membrane and ER. CFTR was detected with
biotinylated mouse monoclonal antibody BJ570 (footnote 3) and
streptavidin Alexa Fluor 488 conjugate (Molecular Probes). The plasma
membrane Ca2+-ATPase was visualized with the mouse
monoclonal antibody 5F10 (Affinity Bioreagents Inc.) and Alexa Fluor
594 goat anti-mouse IgG conjugate (Molecular Probes). B,
C-terminal truncations influence localization of CFTR. CFTR was
detected with the mouse monoclonal antibody BJ570 and visualized with
Alexa Fluor 488 goat anti-mouse IgG conjugate (Molecular Probes).
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Fig. 3.
Pulse-chase experiments showing the turnover
of the immature and mature forms of wild-type and C-terminal
truncations of CFTR. Cells were starved of methionine for 30 min,
pulse-labeled with [35S]methionine (100 µCi/ml) for 15 min, and chased with methionine replete medium for the times indicated
in hours. Immunoprecipitations were preformed as described (15).
Following electrophoresis, gels were dried, exposed to x-ray films, and
then separately subjected to quantitative electronic autoradiography to
enable estimation of the proportion of the initial 35S
radioactivity in the immature band after the pulse, which is present in
either band at subsequent chase times.
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Fig. 4.
A, alignment of C-terminal sequence of
CFTR from different species showing three regions which impact the
stability of the protein. Each of the 3 shaded regions were those
identified by the deletion mutants. In addition, this portion of CFTR
contains two consensus sequences known to play a role in trafficking.
This is a tyrosine-based signal at amino acid position 1424, defined as
YXX*, where * is a hydrophobic amino acid, and a dileucine
(LL) motif at amino acid position 1430/1431. Both sequences are
endocytic sorting signals and mediate protein internalization from the
cell surface (38, 39, 43). B, Western blot showing
requirement of region I (hydrophobic residues 1413-1416) for mature
CFTR stability. The amino acids indicated were replaced by alanine
residues, wild-type CFTR (WT) is shown as a control. 10 µg of whole
cell lysates were separated by 6% SDS-PAGE and analyzed by Western
blotting using the antibody M3A7 (16). C, immunofluorescence
staining showing the absence of CFTR at the cell surface on alanine
substitution of these hydrophobic residues. Immunolocalization was
performed as described in the legend to Fig. 2 using the antibody
BJ570. D, pulse-chase experiments showing that this
substitution causes persistence of the immature form and lack of
appearance of the mature form of CFTR. Pulse-chase experiments were
performed as described in the legend to Fig. 3.
CEHRI) impaired the processing of the protein (Fig. 5D).
Although the relatively high degree of conservation of these five amino
acids may seem surprising if they serve only as a spacer, from the
point of view of protein stability alanines appear to adequately
replace the native sequence.
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Fig. 5.
Role of region II (residues 1400-1404) in
the stability and maturation of nascent CFTR. Rapid turnover of
nascent 1400X CFTR is not influenced by treatment of cells with BFA
(A). Cells stably expressing 1400X CFTR were treated for 90 min with 10 µg/ml BFA and pulse-chase experiments were performed as
described in the legend to Fig. 3. The fast turnover of 1400X CFTR is
inhibited by the proteasome inhibitors lactacystin, ALLN, and NLVS
(B). BFA and proteasome inhibitors were added to cells 90 min prior to methionine starvation to a final concentration of 10 µg/ml (BFA), 10 µM (NLVS), or 50 µM (lactacystin, MG-132, and ALLN) and were present in
all incubations. The amount of [35S] 1400X CFTR was
quantified after a 1-h chase. Alanine substitutions of residues
1400-1404 does not affect CFTR maturation and turnover in BFA-treated
cells (C). Alanine substitutions of all or parts of region
II does not influence the steady state level of nascent or mature CFTR,
however, deletion of this region ( CEHRI) reduces the amount of
mature protein (D). Whole cell lysates of stably transfected
BHK-21 cells (10 µg of protein) were subjected to 6% SDS-PAGE and
Western blotting was performed using M3A7 antibody (16). The indicated
amino acids were replaced by alanine residues.
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Fig. 6.
Alanine substitutions of residues in region
III (residues 1390-1394) is without influence on CFTR maturation.
The specific sequence at residues 1390-1394 (QAFAD) can be replaced by
alanines without affecting CFTR maturation and stability. Pulse-chase
experiments were performed as described in the legend to Fig. 3.
Substitution of residues 1390-1394 (region III) with alanine residues
does not affect CFTR maturation and turnover of the protein in the
presence of BFA.
efflux experiments were performed. Fig.
7 shows that the rates of efflux
following elevation of cellular cyclic AMP levels were as great after
truncation of the final 61 residues (1420X) as with wild-type,
indicating that this portion of the protein is not directly involved in
regulated channel function. Replacement of residues 1413-1416 (FLVI),
however, resulted in only a slight efflux response that was delayed,
consistent with the minimal amount of mature protein formed. A very
similar result was obtained with the 1400X truncation. Interestingly
small but slightly elevated responses were detected with the 1390X and
1380X variants where detectable amounts of mature protein were again
formed. However, the amount of channel activity is considerably less
than would be expected just on the basis of the amounts of mature
protein produced by these two variants. It may not be surprising that truncation so close to NBD2 impairs regulated channel function.
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Fig. 7.
Effect of C-terminal truncations and alanine
substitutions in region I (1413-1416) on chloride channel
activity. Cells grown in 6-well culture dishes were incubated with
loading buffer containing 0.5 µCi of 36Cl
for 1 h at room temperature, washed with efflux buffer, and
stimulated with forskolin-containing buffer (17). Symbols are as
follows: WT (closed circles), 1420X (open
circles), FLVI (open diamonds), 1400X (closed
squares), 1390X (closed triangles), 1380X (open
inverted triangles). The stimulation at time 0 is indicated by an
arrow. Each point represents the average of three
independent samples and standard deviations are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank April Mengos and Tim Jensen for technical assistance, Bradley Bone for antibody production and biotinylation, Susan Bond for preparation of the manuscript, and Marv Ruona for preparation of the graphics.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health, NIDDK Grant DK54076 and a Fellowship from the Deutsche Forschungsgemeinschaft (to M. G.).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.
To whom correspondence should be addressed: Mayo Clinic
Scottsdale, S. C. Johnson Medical Research Center, 13400 E. Shea
Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6206; Fax: 480-301-7017;
E-mail: riordan@mayo.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M003672200
2 X.-B. Chang and J. R. Riordan, unpublished data.
3 T. J. Jensen, B. G. Bone, and J. R. Riordan, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; NBD, nucleotide-binding domain; TMD, transmembrane domain; ABC, adenine nucleotide binding cassette; PDZ, PSD-95, disc-large, ZO-1; BFA, brefeldin A; ALLN, N-acetyl-leucinyl-leucinyl-norleucinal; MG-132, N-carbobenzoxyl-leucinyl-leucinyl-leucinal; NLVS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucinyl-leucinyl-leucinyl-vinylsulfone; PBS, phosphate-buffered saline.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Seibert, F. S., Loo, T. W., Clarke, D. M., and Riordan, J. R. (1997) J. Bioenerg. Biomembr. 29, 429-442[Medline] [Order article via Infotrieve] |
2. |
Akabas, M. H.
(2000)
J. Biol. Chem.
275,
3729-3732 |
3. | Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh, M. J., and Smith, A. E. (1991) Cell 66, 1027-1036[Medline] [Order article via Infotrieve] |
4. | Tabcharani, J. A., Chang, X.-B., Riordan, J. R., and Hanrahan, J. W. (1991) Nature 352, 628-631[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Luo, J.,
Pato, M. D.,
Riordan, J. R.,
and Hanrahan, J. W.
(1998)
Am. J. Physiol.
274,
C1397-1410 |
6. | Gadsby, D. C., and Nairn, A. C. (1999) Physiol. Rev. 79, S77-S107[Medline] [Order article via Infotrieve] |
7. |
Naren, A. P.,
Cormet-Boyaka, E.,
Fu, J.,
Villain, M.,
Blalock, J. E.,
Quick, M. W.,
and Kirk, K. L.
(1999)
Science
286,
544-548 |
8. | Rich, D. P., Gregory, R. J., Cheng, S. H., Smith, A. E., and Welsh, M. J. (1993) Receptors Channels 1, 221-232[Medline] [Order article via Infotrieve] |
9. |
Zhang, L.,
Wang, D.,
Fischer, H.,
Fan, P. D.,
Widdicombe, J. H.,
Kan, Y. W.,
and Dong, J. Y.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10158-10163 |
10. |
Haardt, M.,
Benharouga, M.,
Lechardeur, D.,
Kartner, N.,
and Lukacs, G. L.
(1999)
J. Biol. Chem.
274,
21873-21877 |
11. | Wang, S., Raab, R. W., Schatz, P. J., Guggino, W. B., and Li, M. (1998) FEBS Lett. 427, 103-108[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Short, D. B.,
Trotter, K. W.,
Reczek, D.,
Kreda, S. M.,
Bretscher, A.,
Boucher, R. C.,
Stutts, M. J.,
and Milgram, S. L.
(1998)
J. Biol. Chem.
273,
19797-19801 |
13. |
Ward, C. L.,
and Kopito, R. R.
(1994)
J. Biol. Chem.
269,
25710-25718 |
14. | Rommens, J. M., Dho, S., Bear, C. E., Kartner, N., Kennedy, D., Riordan, J. R., Tsui, L.-C., and Foskett, J. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7500-7504[Abstract] |
15. |
Loo, M. A.,
Jensen, T. J.,
Cui, L.,
Hou, Y.-X.,
Chang, X.-B.,
and Riordan, J. R.
(1998)
EMBO J.
17,
6879-6887 |
16. | Kartner, N., Augustinas, O., Jensen, T. J., Naismith, A. L., and Riordan, J. R. (1992) Nat. Genet.. 1, 321-327[Medline] [Order article via Infotrieve] |
17. | Chang, X., Cui, L., Hou, Y., Jensen, T. J., Aleksandrov, A. A., Mengos, A., and Riordan, J. R. (1999) Mol. Cell 4, 137-142[Medline] [Order article via Infotrieve] |
18. | Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
19. | Saras, J., and Heldin, C. H. (1996) Trends Biochem. Sci. 21, 455-458[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Songyang, Z.,
Fanning, A. S.,
Fu, C.,
Xu, J.,
Marfatia, S. M.,
Chishti, A. H.,
Crompton, A.,
Chan, A. C.,
Anderson, J. M.,
and Cantley, L. C.
(1997)
Science
275,
73-77 |
21. |
Moyer, B. D.,
Denton, J.,
Karlson, K. H.,
Reynolds, D.,
Wang, S.,
Mickle, J. E.,
Milewski, M.,
Cutting, G. R.,
Guggino, W. B.,
Li, M.,
and Stanton, B. A.
(1999)
J. Clin. Invest.
104,
1353-1361 |
22. |
Hall, R. A.,
Ostedgaard, L. S.,
Premont, R. T.,
Blitzer, J. T.,
Rahman, N.,
Welsh, M. J.,
and Lefkowitz, R. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8496-8501 |
23. |
Fanning, A. S.,
and Anderson, J. M.
(1999)
J. Clin. Invest.
103,
767-772 |
24. | Lukacs, G. L., Mohamed, A., Kartner, N., Chang, X.-B., Riordan, J. R., and Grinstein, S. (1994) EMBO J. 13, 6076-6086[Abstract] |
25. | Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129-135[Medline] [Order article via Infotrieve] |
26. | Ward, C. L., Omura, S., and Kopito, R. R. (1995) Cell 83, 121-127[Medline] [Order article via Infotrieve] |
27. | Zhang, F., Kartner, N., and Lukacs, G. L. (1998) Nat. Struct. Biol. 5, 180-183[Medline] [Order article via Infotrieve] |
28. | Chen, E. Y., Bartlett, M. C., and Clarke, D. M. (2000) Biochemistry 39, 3797-3803[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Bogyo, M.,
McMaster, J. S.,
Gaczynska, M.,
Tortorella, D.,
Goldberg, A. L.,
and Ploegh, H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6629-6634 |
30. |
Huang, P.,
Trotter, K.,
Boucher, R. C.,
Milgram, S. L.,
and Stutts, M. J.
(2000)
Am. J. Physiol. Cell Physiol
278,
C417-422 |
31. |
Sun, F.,
Hug, M. J.,
Bradbury, N. A.,
and Frizzell, R. A.
(2000)
J. Biol. Chem.
275,
14360-14366 |
32. |
Sun, F.,
Hug, M. J.,
Lewarchik, C. M.,
Yun, C.,
Bradbury, N. A.,
and Frizzell, R. A.
(2000)
J. Biol. Chem.
275,
29539-29546 |
33. |
Stutts, M. J.,
Rossier, B. C.,
and Boucher, R. C.
(1997)
J. Biol. Chem.
272,
14037-14040 |
34. | Reddy, M. M., Light, M. J., and Quinton, P. M. (1999) Nature 402, 301-304[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Moyer, B. D.,
Duhaime, M.,
Shaw, C.,
Denton, J.,
Reynolds, D.,
Karlson, K. H.,
Pfeiffer, J.,
Wang, S.,
Mickle, J. E.,
Milewski, M.,
Cutting, G. R.,
Guggino, W. B.,
Li, M.,
and Stanton, B. A.
(2000)
J. Biol. Chem.
275,
27069-27074 |
36. | Thiebaut, F., Tsuro, T., Hamara, H., Gottesmann, M. M., Pastan, I., and Willingham, M. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7735-7738[Abstract] |
37. | Cordon-Cardo, C., O'Brien, J. P., Casals, D., Rittman-Grauer, L., Biedler, J. L., Melamed, M. R., and Bertino, J. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 695-698[Abstract] |
38. |
Prince, L. S.,
Peter, K.,
Hatton, S. R.,
Zaliauskiene, L.,
Cotlin, L. F.,
Clancy, J. P.,
Marchase, R. B.,
and Collawn, J. F.
(1999)
J. Biol. Chem.
274,
3602-3609 |
39. |
Weixel, K. M.,
and Bradbury, N. A.
(2000)
J. Biol. Chem.
275,
3655-3660 |
40. |
Sharma, N.,
Crane, A.,
Clement, J. P.,
Gonzalez, G.,
Babenko, A. P.,
Bryan, J.,
and Aguilar-Bryan, L.
(1999)
J. Biol. Chem.
274,
20628-20632 |
41. | Fiedler, K., Veit, M., Stamnes, M. A., and Rothman, J. E. (1996) Science 273, 1396-1399[Abstract] |
42. |
Mickle, J. E.,
Macek, M., Jr.,
Fulmer-Smentek, S. B.,
Egan, M. M.,
Schwiebert, E.,
Guggino, W.,
Moss, R.,
and Cutting, G. R.
(1998)
Hum. Mol. Genet.
7,
729-735 |
43. |
Marsh, M.,
and McMahon, H. T.
(1999)
Science
285,
215-220 |