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(Received for publication, March 24, 1995; and in revised form, June 27, 1995) From the
The Escherichia coli SecB protein binds newly
synthesized precursor maltose-binding protein (preMBP) and promotes its
rapid export from the cytoplasm. Site-directed mutagenesis of two
regions of SecB was carried out to better understand factors governing
the SecB
The interaction of the export chaperone SecB with nascent or
newly synthesized preproteins is an early event in Escherichia coli protein export(1) . SecB binding prevents the premature
folding or aggregation of preproteins and maintains them in an
export-competent
conformation(2, 3, 4, 5) . SecB also
greatly stimulates the rate of protein export, possibly through a
``targeting'' activity that involves conveying preprotein to
the export apparatus in a specific conformation(6) . The best
studied natural SecB ligand is maltose-binding protein (MBP), ( There are
differences between the SecB Mutational studies could be used to identify SecB residues that
function in preprotein binding. Previously, chemically induced amino
acid substitution mutations were shown to cluster in two regions of
SecB(16, 17) . Mutations in region 1 were tightly
clustered at positions Leu-75, Cys-76, and Glu-77 (Fig. 1).
Mutations in the amino-terminal region 2 occurred at the acidic
residues Asp-20 and Glu-24. Two region 1 mutants studied in detail, secBL75Q and secBE77K, are both strongly defective in
promoting the rapid export of MBP from the cytoplasm. However, purified
mutant SecB protein containing either alteration retains the ability to
interact with unfolded MBP and prevent folding in
vitro(16) . These two mutations cause SecB to exhibit
apparently enhanced binding to unfolded MBP, suggesting that region 1
may play a role in preprotein binding. To better understand the roles
of regions 1 and 2 in SecB function, a new set of mutations was
generated in both regions. We describe here the effect these mutations
exert on both the kinetics of MBP export and SecB:preMBP complex
formation.
Figure 1:
Single residue
substitutions in SecB. The topsequence shows the
wild-type SecB amino acid sequence. Columnsbelow represent single residue substitutions at each position. Mutations
were generated using corresponding oligonucleotides containing low
level random base substitutions (see ``Materials and
Methods''). Mutants were divided into 3 classes using a colony
color assay as a semi-quantitative measure of secB activity.
Class I mutants were the least defective in secB activity,
while Class III mutants were extremely defective in secB activity.
To further clarify the roles of region 1 and region 2, we
generated a new collection of single-residue substitution mutants in
both regions and characterized their effects on SecB function. To
circumvent the limitations associated with chemical mutagenesis,
mutations were targeted to region 1 and region 2 using synthetic
oligonucleotides containing low level random base substitutions. To
identify secB mutants, we employed a red/white colony color
assay that monitors the localization of MBP to the periplasm and
thereby reflects cellular secB activity(26) . This
assay relies on the expression of preMBP(18-1), a form of MBP
containing a partially defective signal sequence. Using this assay, a
mutant screen was set up to identify new secB mutants based on
their failure to efficiently complement a chromosomal secBL75Q
mutation. Employing this screen, 20 unique single residue substitutions
were recovered in region 1 (Fig. 1). Small insertion or deletion
mutations, as well as stop-codon mutations, were recovered at other
positions within region 1, indicating that mutagenesis occurred
throughout the targeted region. 10 unique substitutions were identified
in region 2, all occurring at positions Asp-20, Ser-22, and Glu-24 (Fig. 1). A number of double, triple, and quadruple
substitutions were also recovered in region 2. However, each of these
contained at least one change at Asp-20, Ser-22, or Glu-24 (data not
shown). This collection of mutants was divided into three classes by
estimating the levels of secB activity using the red/white
colony color assay (Fig. 1). In these experiments, plasmids
expressing mutant SecB supplied the only source of cellular SecB. Class
I mutants retained substantial secB activity and included all
of the substitutions at Phe-74, Cys-76, Val-78, plus E77D and S22A in
region 2. Class II mutants showed a more severe secB deficiency and included most substitutions at Leu-75 and Glu-77 in
region 1 and all of the substitutions at Asp-20 and Glu-24 in region 2.
Three class III mutants, each the result of proline substitution,
occurred in region 1. These mutants lacked secB activity
completely because they failed to support growth of E. coli on
rich media, a characteristic of E. coli strains devoid of
SecB(27) . Western blot analysis of numerous region 1 and
region 2 mutants revealed that only the proline substitutions in region
1 prevented high-level accumulation of SecB in growing cells (data not
shown). Proline substitution in this region probably prevents normal
folding, leading to rapid proteolytic degradation.
Figure 2:
Kinetics of maltose-binding protein export
in secB mutant strains. secB mutant plasmids were
introduced into strain CK1961 (malE
PreMBP(18-1) was coimmunoprecipitated
with SecB using anti-SecB antisera (Fig. 3A, lane2). Other SecB ligands, such as preLamB and proOmpA, were
not detected because they contain functional signal sequences and are
rapidly exported from the cytoplasm. PreMBP(18-1) did not
fortuitously cross-react with SecB antibodies because it was not
precipitated from extracts lacking SecB due to mutation (Fig. 3A, lane1). Co-precipitation
of SecB and preMBP(18-1) was reduced if 5 µg of unlabeled
purified competitor SecB was added to the extracts prior to the
addition of antisera, indicating that SecB and preMBP(18-1) were
present in these extracts as soluble complexes (Fig. 3B, lanes1 and 3).
SecB
Figure 3:
Detection of SecB:preMBP(18-1)
complexes in radiolabeled cell extracts. Extracts were prepared and
incubated with anti-SecB antiserum or anti-MBP antiserum as described.
Immune complexes were isolated and fractionated by SDS-polyacrylamide
gel electrophoresis; fluorograms are shown. M, anti-MBP
antiserum; B, anti-SecB antiserum. A, extracts were
prepared from [
The results of
co-precipitation experiments with five region 1 mutants are shown in Fig. 3C. Complexes between SecBF74I and
preMBP(18-1) were not detected using either anti-SecB or anti-MBP
antisera (Fig. 3C, lanes1 and 2). Similarly, complexes involving SecBC76Y or SecBQ80R were
observed at greatly reduced levels using either anti-SecB or anti-MBP
antisera (Fig. 3C, lanes5 and 6 and lanes9 and 10). In contrast,
complexes involving SecBL75R or SecBE77V were observed at levels
comparable with that seen with normal SecB (Fig. 3C, lanes3 and 4 and lanes7 and 8). While approximately 12-15% of the total
precipitable preMBP(18-1) was observed in SecB Overall, region 1
mutants showed a distinct pattern of preMBP(18-1) binding defects (Fig. 4). Substitutions at alternating residues Phe-74, Cys-76,
Val-78, and Gln-80 abolished or greatly reduced preMBP binding. In
contrast, diverse substitutions at Leu-75 and Glu-77 all failed to
disrupt preMBP complex formation. There are two exceptions to this
pattern in region 1: SecBF74Y stably bound preMBP(18-1), whereas
three Phe-74 substitution mutants involving the branched chain amino
acids did not, indicating that an aromatic side chain at position 74 is
essential for preMBP binding. Also, SecBV78G formed complexes with
preMBP(18-1) but SecBV78F did not, indicating that only changes
that introduce bulky side chains at Val-78 are sufficient to disrupt
preMBP binding.
Figure 4:
Summary of SecB:preMBP(18-1)
co-precipitation results.
It is striking that mutations at
Phe-74/Cys-76/Val-78/Gln-80 appear to strongly destabilize
preMBP(18-1) binding but result in only mild defects in the rate
of MBP export in living cells. During the course of our binding assay,
preMBP(18-1) may continue to fold into a mature-like form that is
not a substrate for SecB binding(13) . When a competing folding
reaction removes preMBP substrate, binding defects due to lowered
affinity could be greatly magnified, leading to an apparent all-or-none
binding pattern. The high level synthesis of SecB, which occurs in
these strains from multi-copy plasmids, may overcome or suppress export
defects caused by a decreased affinity for preprotein. These results
suggest that the side chains at Phe-74/Cys-76/Val-78/Gln-80 are part of
a hydrophobic preMBP binding pocket, or are required for the formation
of such a binding pocket. The alternating pattern of binding-deficient
mutations further suggests that region 1 is one strand of Intermixed with the binding site residues are Leu-75 and Glu-77. If
region 1 is one strand of Region 2 has several features in common with region 1. First, the
alternating pattern of mutations suggests that region 2 also is
composed of The
overall similarities in mutant phenotypes caused by changes at
Asp-20-Glu-24 and Leu-75-Glu-77 suggests that these
residues function together in a SecB-mediated activity distinct from
preprotein binding. Clues to the roles of Asp-20, Glu-24, Leu-75, and
Glu-77 in SecB function come from previous biochemical studies of SecB
protein altered at two of these positions. Purified SecB protein
carrying the L75Q or E77K substitution retained the ability to interact
with chemically unfolded MBP and prevent its rapid
refolding(16) . In those studies, both mutant SecB proteins
were more effective than normal SecB at blocking the refolding of MBP,
indicating that certain alterations at Leu-75 and Glu-77 result in SecB
with a higher affinity for unfolded MBP. Residues Leu-75 and Glu-77,
together with Asp-20 and Glu-24, may function to facilitate a
conformational change leading to the opening of a closely associated
hydrophobic preprotein binding site. Randall has reported evidence
suggesting that positively charged regions of an unfolded preprotein
are first bound at a site on SecB, and then a conformational change
occurs and a second hydrophobic site on SecB becomes
acccessible(12) . According to this two-step model of
preprotein binding, Leu-75 and Glu-77 mutants could be locked in a
normally short-lived high affinity conformation.
Volume 270,
Number 39,
Issue of September 29, pp. 22831-22835, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
preMBP interaction. 30 aminoacyl substitution mutants
were analyzed, revealing two distinct classes of secB mutants.
Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or
Gln-80 reduced the ability of SecB to form stable complexes with
preMBP, but caused only mild defects in the rate of MBP export from
living cells. The pattern revealed by this class of mutants suggests
that a primary binding site for preMBP is hydrophobic and contains
-sheet secondary structure. In contrast, substitutions at Asp-20,
Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP
export but did not disrupt SecBpreMBP complex formation. These
largely acidic residues may function to regulate the opening of a
preprotein binding site, allowing both high affinity preprotein binding
and rapid dissociation of SecBpreprotein complexes at the
membrane translocation site.
)the periplasmic maltose receptor required by E. coli for maltose utilization(7) . SecB binds to nonoverlapping
regions within a central portion of unfolded MBP(8) . Exported
proteins that do not require the chaperone activity of SecB for export
may interact with the GroEL-GroES or DnaK-DnaJ-GrpE heat shock
chaperones prior to export(9, 10) .preprotein interaction and the
interaction of the heat shock chaperones with their substrates. The
heat shock chaperones have broad substrate-binding specificities, and
they bind and hydrolyze ATP in order to release bound
proteins(11) . In vitro, SecB binds with high affinity
to a variety of peptides and unfolded proteins, many of which are not
natural SecB substrates(12, 13) . SecB is not known to
bind or hydrolyze ATP. Dissociation of SecB preprotein complexes
may involve preprotein binding to SecA, a peripheral membrane protein
required for general protein export. SecA binds and hydrolyzes ATP (14) and, when membrane-bound, has a high affinity for
SecBproOmpA complexes formed in vitro(15) .
Strains and Plasmids
The parent strain used in
this work is the E. coli K-12 derivative MC4100 (F-
lacU169 araD139 rbsR rpsL thiA relA). CK1961 is MC4100 secB::Tn5 malT
zhe::Tn10
recA1. HK51 is MC4100 secB4 malTmalE18-1 srl::Tn10 recA1. HK57
is MC4100 secB::Tn5 malTmalE18-1 srl::Tn10 recA1. pHK202, a
derivative of pBR322, lacks the tet gene and all sequences
between the EcoRI site and the AvaI site and contains
in addition an fd phage ori region for packaging
single-stranded phage DNA. pHK205, the parent plasmid of the mutants
described here, contains the 4.5-kilobase pair EcoRI fragment
from pAK323(18) , carrying the normal secB gene,
cloned into the unique EcoRI site of pHK202.Site-directed Mutagenesis
The 34-mer
oligonucleotide 5`-AAACCGCGTTCCTGTGTGAAGTTCAGCAGGGCGG corresponding to
region 1, and the 40-mer 5`-TTTATACCAAGGATATCTCTTTCGAAGCGCCGAACGCGCC
corresponding to region 2, were synthesized under conditions that
should result in an average of 1.5 mutations/oligonucleotide molecule.
This was accomplished by adding 5.88% (v/v) of an equimolar mixture of
all four nucleotide precursors to each monomer phosphoramidite
solution. Mixing proportions were determined from equations described
by Ner and Smith(19) . Single-stranded templates of pHK205 were
prepared in the dutung
strain CJ236 (20) using R408 helper phage. Phosphorylated mutagenic
oligonucleotides were used to direct DNA synthesis as described
previously(20) , except that T7 DNA polymerase holoenzyme was
used in place of T4 DNA polymerase for the synthesis
reaction(21) . Mutagenized plasmid pools prepared from these
reactions contained greater than 1
10
members.secB Mutant Screen
Mutagenized plasmid pools were
introduced into HK51 by electroporation and plated on tetrazolium
indicator agar (22) supplemented with 1.0% (w/v) maltose and
ampicillin (100 µg/ml). The HK51 screening strain constitutively
synthesizes preMBP(18-1), which contains an arginine substitution
in the hydrophobic core of the signal sequence(23) ,
effectively blocking export of about 95% of total preMBP(24) .
HK51 also contains a partially defective chromosomal secBL75Q
gene, further impairing MBP export to the extent that the strain is
unable to utilize maltose (Mal) and produces red
colonies on maltose tetrazolium indicator agar. The secBL75Q
mutation is efficiently complemented by a cloned wild-type secB gene resulting in partially restored MBP(18-1) export and
Mal
white colonies. Plasmid transformants carrying new secB mutations produced pink or red Mal
colonies on indicator agar due to their failure to complement the
chromosomal secBL75Q mutation. Transformants were screened
directly for pink or red Mal
phenotypes after growth
at 30 °C. In these mutagenesis experiments, approximately
11-15% of total Amp
transformants were
Mal. Mutations were identified by DNA sequencing of
the mutagenized region using Sequenase 2.0 (U. S. Biochemical Corp.)
and procedures and reagents supplied with the enzyme. Prior to DNA
sequencing of the region 2 mutants, extracts of candidate mutants were
first examined for the presence of full-length SecB protein by Western
blot analysis. To confirm that mutagenesis did not introduce other secB mutations, the entire secB sequence of the
following 18 mutants was determined: F74I, F74Y, L75R, L75M, L75P,
C76Y, E77V, E77A, V78F, Q79P, Q80R, D20H, D20A, D20Y, E24Q, E24D, E24A,
E24V. In no case was a second mutation found.
Pulse-chase Analysis of MBP Export
secB mutant plasmids were introduced into CK1961 by electroporation.
Cultures were grown in M63 media (22) supplemented with 0.2%
(v/v) glycerol, 0.4% maltose, thiamine (0.5 µg/ml), MgSO (10 mM), and carbenicillin (100 µg/ml). Pulse-chase
experiments measuring preMBP export were carried out as described
previously(5) . preMBP and MBP bands were quantitated using
phosphorimaging methods, using software provided by the manufacturer
(Molecular Dynamics). The fraction of total MBP in precursor form was
calculated after adjusting for the fact that preMBP contains 9
methionines and mature MBP contains 6 methionines.Preparation of Antisera
His-tagged SecB protein
was purified from extracts of cells expressing a His-tagged secB derivative (
)using procedures and plasmids provided by
the manufacturer (Qiagen). Rabbits were immunized with His-tagged SecB
protein as described previously(25) , except that
Titermax(TM) was used as adjuvant. Anti-SecB antiserum was
preadsorbed with an E. coli cell extract lacking SecB protein
prior to use in immunoprecipitation experiments. Anti-MBP antiserum was
prepared previously(25) .Detection of SecB
secB mutant plasmids were introduced into
HK57 by electroporation. Cultures were grown at 37 °C in M63
minimal media as described above and labeled with 50 µCi of
TranpreMBP(18-1)
ComplexesS-label/ml of culture for 5 min. 1-ml portions were
transferred to chilled centrifuge tubes, and spheroplasts were prepared
using egg white lysozyme (Sigma) as described previously(2) .
Spheroplasts were collected by 30 s of centrifugation at maximum speed
in an Eppendorf model 5415 microcentrifuge at 4 °C. The supernatant
periplasmic fraction was discarded, and the spheroplasts were gently
lysed in 0.5 ml of buffer I (50 mM Tris-HCl, pH 8.0, 150
mM NaCl, 1 mM Na
-EDTA,
2%(v/v) Triton X-100) by repeated pipetting. Protease inhibitor
phenylmethylsulfonyl fluoride was immediately added to a final
concentration of 0.1 mM. Extracts were centrifuged for
30-45 s at maximum speed in an Eppendorf model 5415
microcentrifuge at 4 °C, and 200-µl supernatant portions were
removed and added to new tubes on ice containing 300 µl of buffer I
and anti-SecB antisera or anti-MBP antisera. Immune complexes were
formed for 1 h at 4 °C with constant mixing. To collect immune
complexes, 50 µl of a slurry of protein A-Sepharose CL-4B beads
(Pharmacia Biotech Inc.) (1:1 in buffer I) was added, and mixing was
continued for 30 min. The beads were pelleted by centrifugation for
15-20 s at low speed in an Eppendorf model 5415 microcentrifuge
at 4 °C and washed twice with 0.5 ml of cold high salt buffer (50
mM Tris-HCl, pH 8.0, 1 M NaCl, 1%Triton X-100) and
once with 0.5 ml of 10 mM Tris-HCl, pH 8.0. Bound radiolabeled
proteins were solubilized in Laemmli gel sample buffer. Electrophoresis
on 12.5 or 15% SDS-polyacrylamide gels and fluorography with
diphenyloxazole were carried out as described previously(5) .
Gels were also analyzed using phosphorimaging detection plates. preMBP
and SecB bands were quantitated using a Molecular Dynamics
PhosphorImager. For the SecB
preMBP stoichiometry measurements,
radiolabeling was performed using TranS-label (ICN), which
contains both [
S]methionine and
[
S]cysteine. Calculations were carried out
assuming uniform labeling of all cysteines and methionines.
PhosphorImager data were adjusted for the fact that SecB contains 5
methionine and 4 cysteine residues and preMBP(18-1) contains 8
methionine residues.
MBP Export Defects Associated with secB Mutants
The
colony color assay results indicate that these mutants fail to
efficiently export preMBP(18-1) to the periplasm. The export
defect was also observed by examining the kinetics of normal MBP export
in a subset of secB mutants. The extent and rate of MBP export
was monitored indirectly by measuring the rate of preMBP signal peptide
cleavage in pulse-chase experiments. As shown in Fig. 2A, substitutions in region 1 resulted in a wide
range of kinetic defects in MBP export. The general pattern emerged
that substitutions at Leu-75 and Glu-77 caused a severe slowing of the
rate of MBP export, whereas changes at Phe-74, Cys-76, Val-78, and
Gln-80 showed weak to moderate impairment of MBP export. Mutants F74Y
and V78G, which emerged in our screen as phenotypically weak mutants,
did not reliably show kinetic defects in MBP export (data not shown).
In region 2, diverse substitutions at Asp-20 and Glu-24 all resulted in
severe defects in MBP export, similar to that observed for
substitutions at Leu-75 and Glu-77 (Fig. 2B). Overall,
these results closely matched our mutant rankings obtained using the
colony color assay.
secB::Tn5 recA1). MBP export kinetics were determined
by monitoring the rate of cleavage of the preMBP leader peptide.
Cultures were pulse-labeled for 15 s with Tran
S-label as
described. A pulse sample was taken, and the chase was begun by adding
chloramphenicol (3.4 µg/ml) and unlabeled methionine (0.1 mg/ml)
simultaneously. Chase samples were collected 30 and 60 s after the
addition of chase. Samples were immunoprecipitated with anti-MBP
antiserum and separated by SDS-polyacrylamide gel electrophoresis.
Relative amounts of preMBP and mature MBP were determined using a
Molecular Dynamics PhosphorImager; percent of total MBP in the
precursor form is shown. Experiments were carried out in duplicate, and
each bar represents the average of two
measurements.
SecB
Since the
defect in MBP export could result from the failure of SecB to associate
with preMBP, region 1 and region 2 mutants were surveyed for their
ability to form SecBpreMBP Complex FormationpreMBP complexes in living cells.
SecBpreMBP complexes were previously identified in cell extracts
using immunoaffinity methods (25) . preMBP(18-1) was
chosen as a substrate to examine complex formation because its
defective leader peptide partially blocks preMBP export, leading to
high intracellular preMBP levels that might favor complex formation.
The altered leader peptide of preMBP(18-1) should not influence
complex formation, since SecB binds to a central portion of unfolded
MBP and binding is not affected by the presence of a signal
peptide(8) .preMBP(18-1) complexes were also detected when anti-MBP
antisera was used in place of anti-SecB antisera (Fig. 3A, lane5).
S]methionine-labeled cells
expressing SecB, preMBP(18-1), or both, as indicated by ±
symbols. The relevant genotypes are as follows: lanes1 and 4, secB::Tn5 malE18-1; lanes2 and 5, secB::Tn5
malE18-1 pHK205(secB
); lanes2 and 6, secB::Tn5
malB101 pHK205(secB). B, the secB::Tn5 malE18-1
pHK205(secB
) strain was labeled with
Tran
S-label. Extracts were prepared and immunoprecipitated
as in panelA. For lane3, 5 µg
of purified His-tagged SecB protein was added to the extract prior to
incubation with anti-SecB antiserum. C,
SecB:preMBP(18-1) complex formation in secB mutant
strains. secB mutant plasmids were transformed into the secB::Tn5 malE18-1 strain HK57. The resulting
strains were labeled with Tran
S-label. Extraction and
immunoprecipitation were as in panelA.
preMBP
complexes with normal SecB, most or all of the normal SecB exists in
SecBpreMBP(18-1) complexes (Fig. 3B, lanes1 and 2). Quantitation of these
co-precipitation results yielded estimated SecBpreMBP(18-1)
stoichiometries of 4-5:1 for normal SecB, SecBL75R, and SecBE77V,
indicating that SecBL75R and SecBE77V share the same binding
stoichiometry as normal SecB. Since SecB is probably a
tetramer(28) , the complexes we observe appear to be composed
of one SecB tetramer and one preMBP(18-1) chain. Since each SecB
monomer contains one peptide binding site(12) ,
SecBpreMBP(18-1) complexes may consist of one SecB tetramer
bound at multiple points along one preMBP chain.
-sheet
secondary structure. Region 1 could be one strand of a stable
-sheet, analogous to the large -sheet that forms the floor of
the peptide binding site in class I MHC protein, a protein that binds a
wide variety of peptides(29) . This site may be identical to
the hydrophobic site described by Randall as capable of binding the
fluorescent compound 1-anilinonaphthalene-8-sulfonate(12) . -sheet secondary structure, then the
Leu-75 and Glu-77 side chains would project out onto the side opposite
that displaying the Phe-74/Cys-76/Val-78/Gln-80 side chains. In such an
arrangement, Leu-75 and Glu-77 are unlikely to participate in the same
hydrophobic preprotein binding site. Consistent with this proposal, we
found that diverse substitutions at both sites did not impair complex
formation (Fig. 4). Nevertheless, changes at Leu-75 and Glu-77
resulted in more severe kinetic defects in MBP export than did changes
at the binding site residues Phe-74/Cys-76/Val-78/Gln-80 (Fig. 2). Excluding the proline substitution at Leu-75,
substitutions at Leu-75 and Glu-77 did not prevent high level
accumulation of SecB in cells, indicating that changes at these
positions do not grossly disturb SecB structure. Taken together, these
observations indicate that Leu-75 and Glu-77 are essential for normal
SecB function, but do not directly participate in preprotein binding. -sheet secondary structure. Second, changes at the
acidic residues Asp-20 and Glu-24 did not disrupt preMBP(18-1)
binding but resulted in slowed rates of MBP export similar to those
associated with changes at Leu-75 and Glu-77 in region 1 (Fig. 2). Also, the two glutamyl residues Glu-24 and Glu-77
share the property that neither tolerates the conservative substitution
by aspartic acid. An essential difference between the two regions is
the apparent absence of binding site residues in region 2.
))
We thank Debabrata RayChaudhuri for good advice and
warm collegiality. We also thank Pamela Gannon and Olivera Francetic
for helpful discussions and members of the R. Isberg lab for technical
advice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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