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(Received for publication, September 27, 1994; and in revised form, November
7, 1994) From the
A preproparathyroid hormone allele from a patient with familial
isolated hypoparathyroidism was shown to have a single point mutation
in the hydrophobic core of the signal sequence. This mutation, changing
a cysteine to an arginine codon at the -8 position of the signal
peptide, was associated with deleterious effects on the processing of
preproparathyroid hormone to proparathyroid hormone in vitro.
To examine the biochemical consequence(s) of this mutation, proteins
produced by cell-free translation of wild-type and mutant cRNAs were
used in assays that reconstitute the early steps of the secretory
pathway. We find that the mutation impairs interaction of the nascent
protein with signal recognition particle and the translocation
machinery. Moreover, cleavage of the mutant signal sequence by
solubilized signal peptidase is ineffective. The consequence of this
mutation on processing and secretion of parathyroid hormone is
confirmed in intact cells by pulse-chase experiments following
transient expression of the mutant protein in COS-7 cells. The
inability of the mutant signal sequence, however, to interfere with the
targeting and processing of other secreted proteins does not support
obstruction of the translocation apparatus as the mechanism underlying
the dominant mode of inheritance of hypoparathyroidism in this family. Proteins destined for residence within membranes or for
secretion contain hydrophobic amino-terminal sequences referred to as
signal sequences(1) . These sequences direct the nascent
polypeptides bound to ribosomes to form a functional junction with
rough endoplasmic reticulum (RER) ( The nascent secretory protein is localized to the
endoplasmic reticulum via a targeting apparatus consisting of the
signal recognition particle (SRP) and its membrane-bound receptor on
RER. The SRP binds to the signal sequence as it emerges from the large
ribosomal subunit. This results in a transient delay or even arrest of
translation (3) aimed in preventing premature folding of the
precursor protein. When the SRP-ribosome complex encounters the SRP
receptor or ``docking protein,'' a series of reactions take
place that result in the insertion of the nascent chain into the
translocation site, release of SRP, resumption of translation, and
initiation of translocation. GTP binding and its hydrolysis are
required for these events to take place (4, 5, 6) . As the nascent polypeptide
transverses the translocation channel (7, 8) and
emerges into the lumen of the endoplasmic reticulum, it is modified
further by the signal peptidase enzyme complex that catalyzes the
endoproteolytic cleavage of the signal sequence. Only a small number
of natural mutations in human signal sequences have been reported to
have direct correlation with defective secretion and associated
pathological states(9, 10, 11) . We have
described such a mutation in the signal peptide of one allele of
preproparathyroid hormone (prepro-PTH) gene from a kindred with a form
of familial isolated hypoparathyroidism(9) . This is an
inherited metabolic disorder characterized by hypocalcemia and
hyperphosphatemia resulting from lack of biologically active
circulating PTH, the major calcium-regulating peptide. In this family,
the disorder was inherited as an autosomal dominant trait(12) .
The single point (T to C) mutation changed the codon at position
-8 (signal peptide residues are numbered negatively starting from
the site of cleavage toward the amino terminus) of the signal peptide
of prepro-PTH from cysteine to arginine, thereby disrupting the
hydrophobic core of the signal sequence (Fig. 1). Associated
with this change was a dramatic impairment in the processing of the in vitro translated mutant prepro-PTH protein to pro-PTH by
microsomal membranes(9) .
Figure 1:
Signal peptide
sequence of human prepro-PTH. Amino acids -25 to -1
constitute the signal peptide of wild-type and mutant (single and
double) forms of human prepro-PTH. The 6 residues following the signal
peptidase cleavage site (arrowhead) make up the pro sequence,
and the 84 amino acids of mature PTH follow. Residues -16 to
-5 (underlined) comprise the hydrophobic core of the
signal peptide. The described patient's mutation (Cys
Which step(s) of the early
secretory process is affected by this mutation is not readily evident.
Conceivably it could preferentially affect one or all of the steps
involved, such as binding to SRPs, targeting to the RER, translocation
through the membrane, and proteolytic processing by signal peptidase.
In this report, we have systematically examined each of these steps
using mutant and wild-type forms of in vitro translated
prepro-PTH proteins by assaying their interaction with components of
these various processes. Moreover, the consequence of this mutation on
processing and secretion of PTH was examined in intact cells by
transient expression of the mutant protein in COS-7 cells. Finally, we
have used a co-transfection assay to define the mechanism by which this
specific mutation could cause clinical hypoparathyroidism in the
presence of a second, apparently normal(9) , PTH allele.
Plasmids
pWT83, pSM83, and pDM83, all containing prepro-PTH(1-83)
sequences without the termination codon, were constructed by subcloning
a HindIII-PstI fragment containing the prepro
sequences from plasmids pWT84, pSM84, and pDM84, respectively, into
plasmid SP-PTH(XbaI/Bal31/ClaI/#6)
comprising mature PTH(1-83) sequences without the termination
codon (15) . In order to construct a plasmid encoding a
protein of the same size as prepro-PTH but lacking a functional signal
sequence, NcoI linkers were introduced into the SmaI
site in the polylinker of
SP-PTH(XbaI/Bal31/ClaI/#7) (15) ,
and a 225-base pair HaeIII-HindIII fragment derived
from the same plasmid was ligated into the NcoI site. This
introduced an ATG initiation codon followed by PTH sequences
(51-82)-Ala in place of the signal sequence. This plasmid was then
restricted with ClaI and HindIII and ligated to a DpnI-HindIII fragment isolated from plasmid
SP-PTH(15) , containing sequences encoding PTH(1-84). The
resulting plasmid (pRM84, for random mutant signal sequence), encoded
Met-PTH(51-82)-Ala-Ser-PTH(1-84). To introduce an N-linked glycosylation site into the PTH-coding sequence of
plasmids pWT84 and pSM84, synthetic oligonucleotides, encoding
Ser-Asn-Gly-Ser-Gly-Glu-Gly-Val-Glu-Ser, were ligated into the
unique TaqI site of the prepro-PTH coding sequence (the
underlined sequence indicates the consensus sequence for N-glycosylation). The resulting plasmids, pWT84(G) and
pSM84(G), differed from pWT84 and pSM84, respectively, only by the
insertion of 9 amino acids between residues Ser-17 and Met-18 of the
mature PTH protein.
Figure 2:
Processing of normal and mutant
prepro-PTH. Autoradiogram of [
Figure 3:
Effect of SRP on translation. The cRNAs
for wild-type (pWT84), single mutant (pSM84), double mutant (pDM84),
random mutant (pRM84), and rabbit globin were translated in a wheat
germ cell-free system in the absence (0) or presence of
exogenous SRP (final concentration 0.02 (1) and 0.04 (2) A280 units/ml).
Figure 4:
Translocation-competent binding to
microsomal membranes. Truncated cRNAs missing the termination codon
were transcribed from plasmids encoding wild-type, single mutant, and
double mutant cDNAs and translated in rabbit reticulocyte lysate
system. Translocation-competent binding to microsomal membranes was
assessed by centrifugation of the membranes through either a
physiological salt- or an EDTA-sucrose step cushion. Radiolabeled
proteins in the pellets (A) and corresponding supernatants (B) are displayed.
Figure 5:
Protease sensitivity of membrane-bound
nascent chains. Wild type (pWT84) and single mutant (pSM84) cRNAs were
translated in the rabbit reticulocyte lysate system in the presence
(+) of dog pancreas microsomal membranes. After translation was
completed, reactions were treated with proteinase K (20 µg/ml) with
(+) or without(-) the addition of 1% Triton X-100.
Radiolabeled translation products were immunoprecipitated and analyzed
by SDS-PAGE.
Fig. 6shows that upon translation
of pWT84(G)-transcribed cRNA in reactions supplemented with microsomal
membranes, two translational products with PTH immunoreactivity were
seen that were not present in the absence of membranes. The smaller
product migrated with an apparent molecular weight slightly greater
than that of authentic pro-PTH and was therefore felt to be pro-PTH(G).
The second product, which migrated more slowly than prepro-PTH(G), was
believed to be the glycosylated form of pro-PTH(G). This was confirmed
by treatment with endoglycosidase H which, by removing carbohydrate on
this peptide, shifted its position on an SDS-PAGE gel to that of
pro-PTH(G). Interestingly, prepro-PTH(G) appeared to be processed much
more efficiently by canine microsomal membranes than the unmodified
form of the protein (compare Fig. 2and Fig. 6), and this
may simply reflect the influence of the extended length or the specific
structure of the nascent chain.
Figure 6:
Glycosylation of wild-type and mutant
prepro-PTH(G). Plasmids pWT84(G) and pSM84(G) were transcribed in
vitro and translated in the rabbit reticulocyte lysate cell-free
system in the absence (-M) or presence (+M) of canine microsomal membranes. Translation products
were immunoprecipitated and treated with (+E) or without (-E) endoglycosidase H.
Translation of pSM84(G)-transcribed
cRNA in the presence of membranes resulted in the appearance of three
PTH-immunoreactive products, two of which co-migrated with pro-PTH(G)
and its glycosylated form, while the third was consistent with the
unprocessed prepro-PTH(G) form. Again, the addition of 9 amino acids to
the mature PTH molecule resulted in a more efficient cleavage of the
signal sequence by microsomal membranes as compared with the unmodified
form (see Fig. 2and Fig. 6). Yet, the mutant signal
sequence was once again processed less efficiently than the wild-type
sequence, as indicated by the persistence of the unprocessed mutant
prepro-PTH(G) form in the immunoprecipitated products. Once cleaved,
however, pro-PTH(G) was glycosylated appropriately as confirmed by
treatment of the reaction products with endoglycosidase H. The addition
of carbohydrate to this moiety provides direct and independent evidence
for its translocation, although significantly impaired, across the
endoplasmic reticulum membranes. Furthermore, no larger glycosylated
product was found that might have represented a protein that was
translocated, glycosylated, and yet not cleaved by signal peptidase.
Therefore, the uncleaved mutant prepro-PTH(G) was not delivered to the
glycosylation machinery on the inner surface of the microsomal
membranes.
Figure 7:
Signal peptidase assay. Following
translation of either pWT84- and pSM84- or pWT84(G)- and
pSM84(G)-transcribed cRNAs in a wheat germ extract, signal peptidase
assays were performed by mixing aliquots of translation mixture and
signal peptidase prepared by directly solubilizing canine pancreatic
rough microsomes.
Figure 8:
Expression of wild-type and mutant forms
of prepro-PTH in COS-7 cells. Plasmids pWT84, pSM84, and pDM84 were
transiently transfected into COS-7 cells. Five days following
transfection, the cells were pulse-labeled with
[
WT52 was co-transfected
into COS-7 cells with either wild-type or mutant prepro-PTH expression
vector. Over-expression of the mutant precursor for up to 10 days did
not interfere with the processing of prepro-PTH(1-52) (Fig. 9). Although these results may simply reflect lack of
sensitivity of the system, they do not support global interference with
protein processing at the microsomal membrane level as a consequence of
overexpression of the mutant prepro-PTH form.
Figure 9:
Co-transfection of wild-type and mutant
prepro-PTH. Plasmids pWT84 and pSM84 were transfected into COS-7 cells
either alone or in combination with pWT52. After the indicated number
of days posttransfection, cells were pulse-labeled with
[
Translocation from the cytoplasm into the endoplasmic
reticulum, is a multistep process requiring a functional amino-terminal
signal peptide. A signal sequence must perform effectively several
distinct functions required for the efficient translocation of secreted
proteins. These subfunctions include its recognition and binding to
SRP, its interaction with membrane-bound components of the export
machinery, opening the protein-conducting channels to initiate
translocation, and appropriate presentation to the signal peptidase for
cleavage. Three domains have been identified as a common feature of
eukaryotic signal sequences, and considered to be necessary for
carrying out these functions: a positively charged NH Two reported inherited
mutations in the signal sequence of human secreted proteins, namely
preprovasopressin(10) , and preprofactor X (11) ,
involve the COOH-terminal region of the respective signal peptides.
Thus, a point mutation resulting in substitution of Arg for Gly at the
-3 position of the factor X signal peptide (Factor X The present study is the first to
examine the effect of a naturally occurring substitution at the
hydrophobic core of a signal peptide that results in human disease.
Prepro-PTH, the precursor of PTH, contains a typical 25-residue
amino-terminal signal sequence followed by a 6-residue prospecific
peptide and the mature hormone (residues 1-84; see Fig. 1). The hydrophobic core of the human prepro-PTH signal
peptide is composed of 12 contiguous uncharged amino acids (residues
-5 to -16 of the signal peptide). In the present study,
we have demonstrated that substitution of a charged amino acid, Arg for
Cys, in the signal peptide hydrophobic core of prepro-PTH impairs
co-translational translocation as well as posttranslational cleavage by
isolated signal peptidase. The impairment was even more evident when
two charged residues were introduced in the hydrophobic core of the
signal sequence. In contrast to deletion mutants(26) , this
change interferes not only with translocation and cleavage by signal
peptidase but also with binding to SRP. Since substitution of a
hydrophobic amino acid, leucine, for cysteine or its deletion was
ineffective in modifying translocation and processing (26) ,
the present findings would suggest that a change in the hydrophobicity
of the core is responsible for the observed global disruption in the
processing of the mutant prepro-PTH. Similarly, single charged amino
acids introduced in the hydrophobic core of the E. coli maltose binding protein signal peptide impair secretion of the
protein into the external periplasmic compartment of the
cell(27) . It is rather intriguing that three distinct
reports of inherited mutations in the signal sequence of human secreted
proteins, namely prepro-PTH(9) ,
preprovasopressin(10) , and preprofactor X(11) ,
demonstrate inheritance of the associated disorder in an autosomal
dominant fashion. As is the case for the other two reported disorders,
however, it remains unclear why individuals with a mutated PTH allele
have hypoparathyroidism. From transfection studies in COS-7 cells, it
would appear that expression of the mutant allele does lead to
secretion of PTH, albeit inefficiently. Moreover, the normal allele
would be expected to produce sufficient circulating PTH to maintain
calcium homeostasis. The only other reported case of familial isolated
hypoparathyroidism segregating with a mutation in the PTH gene,
involved a point mutation affecting intron splicing and was associated
with autosomal recessive inheritance of the disorder(28) .
Since heterozygous individuals for this mutant allele were unaffected,
it would appear that one normal PTH allele is sufficient for
maintaining calcium homeostasis. Hypopathyroidism in the presence of
one normal PTH allele would therefore suggest that the mutant gene
product exerts a dominant negative effect in vivo. The mutant
protein might interfere with the normal targeting and processing of
other secreted proteins, including the normal PTH precursor. Such
interference might even lead to destruction of parathyroid tissue in
affected individuals; unfortunately, this is difficult to evaluate
because the tissue is not readily accessible. Export incompatibility,
however, has been observed in E. coli expressing
transport-defective The phenotype of the
single-mutant prepro-PTH suggests that it might have dominant negative
effects under appropriate conditions. The mutation allows a fraction of
the prepro-PTH precursor to enter the translocation machinery, but the
mutant protein is then cleaved inefficiently by signal peptidase. The
inability of the uncleaved protein to reach the glycosylation machinery
(assessed using the precursor modified by inclusion of a glycosylation
signal) suggests that the precursor is not transported fully across the
microsomal membrane. Such a protein that engages the translocation
apparatus but fails to move through the apparatus efficiently might
well have dominant negative effects. The competition experiment in Fig. 9failed to demonstrate such an effect; perhaps higher
levels of protein expression or a longer term experiment are needed.
Nevertheless, the observation that all three reported human signal
sequence mutations involve proteins that partly engage the secretory
apparatus and appear to have dominant effects suggests that in vivo these mutant proteins cause dominant secretory dysfunction.
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1629-1635
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)membranes, thereby
assuring the translocation of the growing polypeptide chain into the
lumen of the endoplasmic reticulum and its subsequent cleavage by the
luminal-localized signal peptidase enzyme (for review, see (2) ).
Arg
at the -8 position; single mutant) and the additional
substitution at the -10 position of the signal peptide (Ala
Arg; double mutant), are indicated by boldface. Numbersabove the amino acids indicate their position relative to
the signal peptidase cleavage site.
Materials
Restriction endonucleases, Klenow
fragment of Escherichia coli DNA polymerase I, and T4 DNA
ligase were purchased from New England Biolabs (Beverly, MA).
Endoglycosidase H (endo-
-N-acetylglucosaminidase H) was
from Boehringer Mannheim. Rabbit reticulocyte lysate, wheat germ
extract, and canine pancreatic rough microsomes were purchased from
Promega Corp. SRPs prepared from freshly excised canine pancreas and
eluted from an aminopentylagarose column (13) were a generous
gift from Reid Gilmore, University of Massachusetts, Worcester, MA.Construction of Plasmids
Plasmid pWT84 was
prepared by inserting a DdeI-XbaI fragment of human
prepro-PTH cDNA encoding prepro-PTH(1-84) within the HindIII-XbaI cloning sites of the mammalian
expression vector pCDM8(9) . The single point mutation (T to C)
was introduced at the -8 position of the signal peptide of
prepro-PTH cDNA using oligonucleotide-directed
mutagenesis(14) , resulting in plasmid pSM84 (single mutant;
see Fig. 1). Plasmid pSM84 was then further modified by two
additional point mutations introduced into the -10 position of
the signal peptide (GCA to CGA; Ala to Arg),
resulting in plasmid pDM84 (double mutant; Fig. 1).In Vitro Transcription, Translation, and Analysis of
Products
Plasmids were linearized and sense RNA strands were
transcribed using either T7 or SP6 RNA polymerase(15) .
Translation reactions in rabbit reticulocyte lysate and in wheat germ
extract were performed according to the manufacturer's (Promega)
procedure. Translation products were immunoprecipitated using
affinity-purified goat anti-human PTH(1-34) antiserum and
subjected to 15-20% continuous gradient SDS-polyacrylamide gel
electrophoresis (PAGE). Autoradiography was performed after treating
the gel with EN
HANCE (DuPont NEN) as described
previously(15) . The identity of PTH-related peptides was
established by amino-terminal radiosequence analysis(16) .Posttranslational Membrane Binding
Truncated cRNAs
missing the termination codon were transcribed from plasmids pWT83,
pSM83, and pDM83 and translated in the rabbit reticulocyte lysate
cell-free system for 15 min at 24 °C. Aurintricarboxylic acid was
added to 0.1 mM to inhibit translation initiation, and after
another 15-min incubation, emetine was added (1 mM final
concentration) to block peptide elongation. Incubation was continued
for another 15 min, 4 eq (1 eq refers to 1 µl of the original rough
microsome preparation that has been adjusted to a concentration of 50 A units/ml; see (17) ) of microsomal
membranes/25 µl of translation mixture were added, and incubation
continued for 15 min at 24 °C. Translation products were then
incubated at 0 °C for 10 min. Insertion into the membranes was
assessed by centrifugation of the membranes through either a
physiological salt- or an EDTA-sucrose step
cushion(15, 18) . Supernatants containing
membrane-free peptides and pellets were immunoprecipitated and
subjected to SDS-PAGE analysis, as described (15) .
Protease Protection Assay
To assay for
co-translational translocation, wild-type and single-mutant cRNAs were
translated in rabbit reticulocyte lysate cell-free system in the
presence of canine pancreas microsomal membranes. After a 15-min
incubation at 24 °C, proteinase K (20 µg/ml final
concentration) was added to the translation reaction mixtures either
alone or in combination with Triton X-100 (1%), and incubation was
continued for a further 60 min on ice. After inactivation of the
protease with phenylmethylsulfonyl fluoride (final concentration, 2
mM), radiolabeled proteins were subjected to
immunoprecipitation and SDS-PAGE analysis.Endoglycosidase H Digestion
Transcribed products
of pWT84(G) and pSM84(G) were translated in the absence or presence of
microsomal membranes, immunoprecipitated, and treated with
endoglycosidase H (10 milliunits) as described previously(19) .
Following overnight digestion, bovine serum albumin was added as a
carrier (final 0.25%), and the samples were precipitated by ice-cold
trichloroacetic acid (15% final concentration). Following
centrifugation, the precipitates were resuspended in 0.1 M Tris-HCl, pH 7.4, and prepared for SDS-PAGE analysis as usual.Signal Peptidase Assay
EDTA-stripped,
nuclease-treated rough microsomes were prepared from canine pancreas as
described(17) . Aliquots of rough microsomes were resuspended
by homogenization in ice-cold buffer (20 mM HEPES, pH 7.6, 50
mM NaCl) to a final concentration of 50 A units/ml. Solubilization of signal peptidase with sodium
deoxycholate was performed as described previously(20) .
Briefly, one volume of 10% (w/w) sodium deoxycholate was mixed with 19
volumes of membrane suspension. The resultant clear solution was
centrifuged at 100,000
g for 4 h, and the supernatant
was frozen in liquid nitrogen. Wild-type and single-mutant prepro-PTH
and preproPTH(G) cRNAs were translated in a wheat germ cell-free
system, and translation products were used as substrates for
detergent-solubilized signal peptidase(20) . A typical
50-µl posttranslational cleavage assay contained 20 µl of the
detergent extract, 20 µl of wheat germ translation mixture, and 10
µl of water. Posttranslational cleavage by signal peptidase was
allowed to proceed at 25 °C for 90 min.Pulse-Chase Analysis
COS-7 cells cultured in
12-well (22-mm diameter) plates in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and
antibiotics were transfected with plasmids pWT84, pSM84, and pDM84
using the DEAE-dextran protocol followed by 10% dimethyl sulfoxide
shock(21) . Five days later, cells were rinsed twice with
Dulbecco's modified Eagle's medium without methionine and
supplemented with 2% dialyzed fetal calf serum and then incubated for
15 min at 37 °C in the same medium containing
[S]methionine (40 µCi/well). The cells were
then washed once with 2 ml of Dulbecco's modified Eagle's
medium (with methionine, supplemented with 10% fetal bovine serum), and
then incubated further in the same medium for the times indicated.
Conditioned media were saved for immunoprecipitation, and cells were
lysed with 0.5 ml of lysis buffer (0.01 M Tris-HCl, pH 7.2, 1%
Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA,
0.02% sodium azide, 0.15 M NaCl) with 50 µl of
phenylmethylsulfonyl fluoride at a final concentration of 500
µg/ml. DNAs of the cell lysates were sheared with a 22-gauge
needle, and the extracts were briefly centrifuged prior to
immunoprecipitation.
Competition Studies
COS-7 cells were transfected
with pWT84 or pSM84 either alone or in combination with pWT52 encoding
for human prepro-PTH(1-52)(22) . After the indicated
times post-transfection, cells were labeled with
[S]methionine for 15 min at 37 °C, as
described above. Cells were then lysed and processed for
immunoprecipitation and SDS-PAGE analysis.
Processing of Wild-type and Mutant prepro-PTH
To
assess the processing of wild-type and mutant forms of prepro-PTH to
pro-PTH, transcribed sense RNA strands were translated in a rabbit
reticulocyte lysate cell-free system in the absence or presence of
increasing concentrations of canine pancreatic microsomal membranes. In
this co-translational assay, substitution of Cys Arg at the
-8 position of the prepro-PTH signal peptide (single mutant)
resulted in impaired processing to pro-PTH as compared with the
wild-type form (Fig. 2). The addition of a second charged
residue within the hydrophobic core of the signal peptide (Ala
Arg at the -10 position, double mutant) completely abolished its
proteolytic processing by rough microsomes. Maximal processing of
wild-type and single-mutant precursors occurred when microsomal
membranes were added to a final concentration of 4 eq/25 µl of
translation mixture.
H]leucine-labeled
proteins derived from the translation of cRNAs transcribed from
plasmids containing wild-type (pWT84), single mutant (pSM84), and
double mutant (pDM84) prepro-PTH cDNA. Translation was performed in the
rabbit reticulocyte lysate cell-free system in the absence (0 eq) or
presence of increasing amounts (4, 8, and 12 eq) of canine pancreatic
microsomal membranes.
Interaction with SRP
The impaired processing of
the mutant prepro-PTH proteins by microsomal membranes may result from
the inability of SRP to recognize efficiently the altered signal
peptides. Binding of SRP to the signal sequence of nascent secretory
proteins induces a site-specific elongation arrest of
translation(3, 23) . We, therefore, assessed the
interaction of the mutant prepro-PTH signal sequences with SRP by
examining the effect of exogenous SRP on inhibition of translation in a
wheat germ cell-free system (Fig. 3). The mRNAs for pWT84,
pSM84, pDM84, pRM84, and rabbit globin were translated in the presence
(final concentration 0.02 and 0.04 A units/ml)
or absence of exogenous SRP. The translation of the wild-type protein
was inhibited significantly more than the translation of the mutant
peptides by exogenous SRP. Moreover, translation of the single-mutant
form was impaired to a greater extend than that of the double mutant.
The addition of SRP did not affect the synthesis of globin (cytoplasmic
protein with no signal peptide) and PTH-like protein with a random
mutant signal peptide. These results suggested that the observed
inhibition in processing of the mutant precursors is, at least, partly
due to the inability of their signal sequences to interact effectively
with SRP.
Translocation-competent Binding
To assess binding
of the mutant prepro-PTH proteins to microsomal membrane components,
truncated cRNAs for wild-type and mutant forms of prepro-PTH missing
the termination codon were transcribed from plasmids pWT83, pSM83, and
pDM83 and translated in the rabbit reticulocyte lysate cell-free
system, thereby ``freezing'' the nascent proteins on
ribosomes. After translation was complete, rough microsomes were added,
and insertion into membranes was assessed following centrifugation of
the reaction mixture through either a physiological salt- or an
EDTA-sucrose step gradient. Only nascent chains tightly bound to the
translocation apparatus pellet with the membranes in the presence of
high concentrations of EDTA(18) . When membranes were added to
the reaction mixture post-translationally, the propeptide (wild-type
and single mutant) as well as the prepropeptide bands were seen in the
precipitates (Fig. 4A). Moreover, peptides pelleting
with the rough microsomes were resistant to extraction with EDTA,
suggesting that translocation-competent binding of the ribosome-nascent
chain complex to the membranes had taken place. This post-translational
assay suggested that the mutant proteins can be targeted to microsomal
membranes and bind in a translocation-competent manner. Although the
mutant forms were able to engage the translocation apparatus, they did
so less efficiently than the wild-type form. Equal loading of the
various reaction mixtures was verified by examining corresponding
supernatant fractions using SDS-PAGE analysis (Fig. 4B). Thus, the amount of nascent protein that
sedimented with rough microsomes paralleled the affinity of SRP for the
respective peptides (wild-type > single mutant > double mutant)
likely reflecting their degree of interaction with SRP. As shown in Fig. 4A, processing of the mutant forms to pro-PTH was
again markedly impaired when compared with the wild-type protein, even
though translocation-competent binding had been achieved. These results
suggested that additional processes in the early steps of the secretory
pathway, i.e. translocation and/or interaction with signal
peptidase, may be impaired by the mutant signal sequences.
Protease Sensitivity of Membrane-bound Nascent Chains
To determine the location of the nascent chains within the
rough microsomes, we examined their sensitivity to digestion with
proteinase K either in the absence or presence of Triton X-100. Protein
products that are translocated to the lumen of the microsomal vesicles
would be protected from digestion by proteolytic enzymes that are
unable to enter these vesicles. As shown in Fig. 5, the
processed peptides (pro-PTH) were protected by the membranes from
proteolysis by proteinase K. The protection of these forms must have
resulted from their sequestration into the lumen of the microsomal
vesicles. This was confirmed by the addition of Triton X-100 to the
reaction mixture, thereby, permeabilizing the membrane bilayer and
allowing the protease to gain access to all protected polypeptides. The
minor unprocessed single-mutant product that is protected from
proteolysis may represent unprocessed nascent protein that has not
fully translocated, yet is protected from proteolysis by the ribosomes
and the tight ribosome-membrane junction required for translocation.
N-Glycosylation of a Modified PTH Sequence
Since
the mutant signal sequence might be a poor substrate for signal
peptidase, cleavage alone is an insufficient criterion for the
localization of PTH peptides. The fact that a fraction of the mutant
prepro-PTH was protected from proteolysis raises the question of how
far into the translocation process the uncleaved precursor has
progressed. Because glycosylation is restricted to the lumen of the
endoplasmic reticulum, the addition of carbohydrate to the PTH sequence
by microsomal membranes would constitute further evidence of
translocation and would not directly require the presence of a suitable
substrate for signal peptidase(19) . Because the mature PTH
protein does not have an N-linked glycosylation site, we
engineered such a site 23 amino acid residues distal to the signal
peptidase cleavage site.
Processing by Solubilized Signal Peptidase
To
determine whether the single-mutant form of prepro-PTH is an unsuitable
substrate for signal peptidase, we assessed proteolytic processing of
the nascent protein in a translocation-independent assay. Following
translation of wild-type and single-mutant prepro-PTH cRNAs in the
wheat germ cell-free system, signal peptidase assays were performed by
mixing aliquots of translation mixture and signal peptidase prepared by
directly solubilizing canine pancreatic rough microsomes. In this
post-translational assay, no cleavage was detected for the mutant
precursor, while a minor amount of processing was observed for the
wild-type form of prepro-PTH (Fig. 7). Since the wild-type
prepro-PTH is also an inefficient substrate for solubilized signal
peptidase, an attempt was made to increase the sensitivity of the assay
using as substrate the modified form of the prepro-PTH molecule
containing an engineered glycosylation target site. RNAs encoding the
wild-type and single-mutant form of prepro-PTH(G) were translated, and
solubilized signal peptidase was added posttranslationally (Fig. 7). As found with intact membranes, wild-type and mutant
forms of this modified PTH protein were processed more efficiently than
their normal counterparts. Indeed, the mutant prepro-PTH(G) was
processed by solubilized signal peptidase unlike its unmodified form,
but the extent of cleavage was significantly less than that of the
wild-type prepro-PTH(G). These results suggested that the mutation in
the signal peptide makes the protein a less suitable substrate for
signal peptidase.
Processing and Secretion of Prepro-PTH in COS-7
Cells
To confirm the cell-free data outlined above in intact
cells, we examined the processing and secretion of mutant and wild-type
forms of prepro-PTH from COS-7 cells transfected transiently with the
corresponding expression plasmids. Five days following transfection,
cells were pulse-labeled for 15 min with
[S]methionine and then chased for the times
indicated in Fig. 8with medium containing cold methionine. The
PTH species in cell extracts and conditioned media were
immunoprecipitated and then resolved by SDS-PAGE. At the earliest time
point examined (0 min), the predominant product immunoprecipitated in
wild-type PTH-transfected cells was pro-PTH, while only barely
detectable levels of prepro-PTH precursor were observed (Fig. 8A). Moreover, immunoreactive PTH was seen in the
culture medium of these cells after 30 min of chase (Fig. 8B). The rate of processing seen with the
wild-type sequence was in striking contrast to that observed with the
mutant forms. In lysates from cells transfected with either mutant, the
predominant band at the earlier time point corresponded with the
prepro-PTH precursor with proteolytic cleavage to pro-PTH being
dramatically reduced in efficiency. With the single mutant, only a
small amount of pro-PTH was detected; no pro-PTH was found in cells
expressing the double mutant. With the single-mutant form, a band
corresponding to prepro-PTH was detectable even after 120 min of chase,
consistent with the in vitro observed inefficient cleavage of
this precursor to pro-PTH by microsomal membranes. Immunoreactive PTH
was detectable in culture media of these cells after 30 min of chase
but at substantially reduced levels as compared with the wild-type
form. The double mutant form of prepro-PTH, although efficiently
translated, was not processed to pro-PTH, nor was immunoreactive PTH
detectable in the culture media of cells transfected with this plasmid
(data not shown). These results demonstrate in intact cells the
inefficient processing of the mutant prepro-PTH molecules as compared
with the wild-type form. The single-mutant prepro-PTH allows a small
amount of normal processing, consistent with the results in the
cell-free studies that showed that the single mutant molecule can
engage the translocation apparatus, although less efficiently than
normal. The double mutant prepro-PTH almost totally fails to engage the
translocation apparatus in cell-free extracts and is not processed at
all in intact cells.
S]methionine for 15 min. At the indicated times
following pulse-labeling, the media were removed, and cell extracts
were prepared. Both cell extracts (A) and media (B)
were immunoprecipitated using a PTH-specific antibody prior to SDS-PAGE
analysis. *, sample not processed.
Competition Experiments
A co-transfection assay in
COS-7 cells was used to address the issue of dominant transmission of
hypoparathyroidism in the family carrying the single-mutant prepro-PTH
allele. The objective was to determine whether the inefficient
processing of the mutant precursor can result in obstruction of the
translocation apparatus and, thereby, to global defects in protein
processing. For this study, an expression vector containing sequences
encoding a truncated version of prepro-PTH, missing the last 32
residues (WT52), was used to provide a readily distinguishable varient
of prepro-PTH with a normal signal sequence.
S]methionine for 15 min. Cell extracts were
then prepared and immunoprecipitated prior to SDS-PAGE
analysis.
terminus, a central hydrophobic core of 10-15 amino acid
residues, and a polar COOH-terminal region(24, 25) .
While the COOH-terminal region influences the efficiency and fidelity
of signal peptidase cleavage, intactness of the hydrophobic region is
indispensable for initiating translocation.) blocks cleavage by signal peptidase but does not
interfere with targeting and translocation to the RER(11) .
Similarly, a naturally occurring substitution of Thr for Ala at the
-1 position of the signal peptide of preprovasopressin results in
central diabetes insipidus(10) . This mutant protein, similar
to the Factor X
, undergoes inefficient cleavage
by signal peptidase, although targeting and translocation to the RER
are not measurably affected.
-galactosidase leading to lethal jamming of
the cellular export machinery(29) .
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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