(Received for publication, April 14, 1995)
From the
Signal peptidase removes amino-terminal signal peptides from
precursor proteins during or immediately following their translocation
to the lumen of the endoplasmic reticulum (ER) and may participate in
ER degradation, a poorly defined process whereby abnormal proteins are
rapidly degraded early in the secretory pathway. Here, the involvement
of signal peptidase in ER degradation is examined through the use of
two chimeric membrane proteins that lack amino-terminal signal
peptides: A189invHD, which contains sequences derived from arginine
permease and histidinol dehydrogenase, and AHDK2, containing the
ER-resident protein Kar2p fused to the carboxyl terminus of A189invHD.
Degradation of approximately 95% of A189invHD is observed in yeast
cells expressing enzymatically active signal peptidase, whereas only
60% undergoes rapid degradation in a sec11 mutant bearing a
temperature-sensitive mutation in the gene encoding the 18-kDa subunit
(Sec11p) of the signal peptidase complex. AHDK2 is proteolyzed in a
reaction yielding at least two fragments in wild-type cells and in the sec11 mutant containing a plasmid bearing the SEC11 gene. The proteolytic reaction is catalyzed in a
temperature-dependent manner in the sec11 mutant, with AHDK2
remaining stable at the nonpermissive temperature. Using conditional
mutants defective in protein translocation into and out of the ER and in vitro protease protection studies, the site of degradation
for AHDK2 is localized to the ER lumen. The data therefore indicate (i)
A189invHD is degraded through both signal peptidase-dependent and
independent processes; (ii) signal peptidase, specifically the Sec11p
subunit, is required for the proteolysis of AHDK2; and (iii) the Kar2
fragment at the carboxyl terminus of AHDK2 permits detection of
proteolytic intermediates.
During or immediately following the translocation of
pre-proteins across the ER
The signal peptidase complex
(SPC) purified from the yeast Saccharomyces cerevisiae is
comprised of four protein subunits with molecular masses of 25, 20, 18,
and 13 kDa(36) . The SEC11 gene encodes the 18-kDa
subunit (Sec11p)(4) . The SPC isolated from canine pancreas
contains five subunits with molecular masses of 25, 23/22, 21, 18, and
12 kDa(9) . An enzymatically active complex containing only two
subunits (23 and 19 kDa) has been isolated from hen
oviduct(2) , suggesting that the catalytic site may be confined
to a subset of the subunits present in the mammalian and yeast SPCs.
Yeast Sec11p is homologous to the 18- and 21-kDa subunits of the
mammalian SPC(16, 27) , although the significance of
two Sec11p homologues in mammals is not understood.
Recent studies
suggest that signal peptidase may have a role not only in the
processing of amino-terminal signal peptides but also in the rapid
degradation of some abnormal proteins within the ER through a process
generally referred to as ER degradation(19, 37) . When
uncomplexed with the H1 subunit of the asialoglycoprotein receptor, H2
is degraded resulting in the accumulation of a 35-kDa proteolytic
intermediate(1, 37) . Amino acid sequencing of the
cleavage site of this intermediate and surrounding residues reveals a
primary sequence organization similar to that of amino-terminal signal
peptides(37) . A dependence on small neutral or small polar
residues for efficient cleavage of H2, identical to that seen for the
processing of signal peptides, is observed following substitutions with
large or charged amino acids at the cleavage site. Though these
substitutions prevent the production of the 35-kDa fragment, they do
not prevent proteolytic elimination of H2. These findings demonstrate
H2 is degraded by at least two pathways, one of which may involve
signal peptidase. In addition, in vitro studies have shown
that a precursor protein derived from the Semliki Forest
virus(20) , mutant forms of the H1 subunit of the
asialoglycoprotein receptor(26) , and the invarient chain of
the major histocompatibility antigen (21) are cleaved near
their transmembrane segments at sequences bearing similarities to
signal peptide cleavage sites.
Due to the absence of specific
pharmacological inhibitors, the involvement of signal peptidase in ER
degradation has not been conclusively demonstrated. In the present
study, we have employed a genetic approach to more directly examine the
role of signal peptidase. Using the sec11 mutant, we
demonstrate that A189invHD, a chimeric membrane protein containing the
cytoplasmic enzyme histidinol dehydrogenase fused to a luminal domain
of arginine permease, is degraded in a manner dependent on functional
signal peptidase. Degradation of AHDK2, a derivative of A189invHD
containing Kar2p (an ER resident protein) fused at its carboxyl
terminus, is blocked in the sec11 mutant at the nonpermissive
temperature, whereas partial degradation is observed at more permissive
temperatures. Complementation of the sec11 mutation through
the introduction of a plasmid encoding wild-type Sec11p into the sec11 mutant results in the cleavage of AHDK2 at, minimally,
two internal sites. Cleavage of AHDK2 and the production of distinct
intermediates are also observed in wild-type (SEC11) cells.
Furthermore, through both genetic and biochemical analyses we identify
the ER lumen as the site of proteolytic processing of AHDK2. The data
presented thus provide strong evidence that signal peptidase can cleave
internal sites within polypeptide chains and participates in an
intra-ER degradative pathway.
Figure 1:
Schematic presentation of protein
chimeras used in this study. The specific sequences comprising
A189invHD have been described earlier(15) . It contains 189
amino acids from the amino terminus (N) of arginine
permease(17) , 134 internal amino acids from invertase (inv)(33) , and, at the carboxyl terminus (C), amino acids 33-799 of HD, a cytoplasmic
polypeptide containing histidinol dehydrogenase(7) . AHDK2 (see
``Experimental Procedures'') differs from A189invHD in that
residues 593-799 of HD are replaced with residues 28-682 of
Kar2p, a resident protein of the ER(22, 25) . The
fragments comprising A189invHD and AHDK2 are not depicted to scale.
Anti-Kar2p and anti-HD antibodies used in this study were raised to the
carboxyl-terminal 216 residues of Kar2p (25) and residues
33-241 of HD(15) ,
respectively.
Preparations of extracts derived from
radiolabeled cells were prepared essentially as described
previously(10, 15) . Cells were sedimented in a
microcentrifuge and then resuspended in 0.2 ml of 10% trichloroacetic
acid. Glass beads were added to the Eppendorf tube in an amount that
produced approximately equal volumes of liquid and glass beads. The
cells were lysed by a 30-s pulse of vortex mixing followed by 30 s of
incubation on ice, repeated 3 times. The mixture of proteins and broken
cells was sedimented in a microcentrifuge for 2 min at 12,000 rpm, and
the pellet was mixed with SDS-PAGE sample buffer (20 µl) and boiled
for 5 min. The boiled proteins were resuspended in a solution (0.7 ml)
containing phosphate-buffered saline/Triton X-100 (1%) and protease
inhibitors. Cell debris was sedimented in a microcentrifuge for 2 min
at 12,000 rpm. Immunoprecipitation was then performed through the
addition of appropriate antiserum, anti-Kar2p (0.5 µl/1 A
For Western blotting,
the TCA-precipitated proteins prepared from cells broken with glass
beads were resolved by SDS-PAGE, blotted onto nitrocellulose, and
detected with either anti-Kar2p (1:5000) or anti-invertase (1:500)
antibodies using alkaline phosphatase-labeled secondary antibodies
(from Bio-Rad).
Autoradiograms depicting bands from pulse-chase
analyses were analyzed on a model DU 70 spectrophotometer (480 nm
wavelength) using a gel scanner adaptor (Beckman Instruments,
Fullerton, CA). A plot of optical density versus position
along the the autoradiogram was obtained. The areas under the peaks
corresponding to bands on the autoradiogram were measured by
programming provided by the manufacturer. Protein levels were recorded
as percent remaining in the chase relative to that present after the
pulse.
For proteinase K protection
analysis, cells of wild-type strain FC2-12B (SEC11)/pAHDK2 (12 mls) were grown to A
As hybrid proteins have been instrumental in
the study of protein degradation within the ER (5, 31) and other compartments(34) , we
reasoned this chimera may be useful for studying the role of signal
peptidase in ER degradation. For this purpose, we employed yeast
strains containing one or both of two temperature-sensitive mutations: sec11, which inhibits signal peptidase activity(4) ,
and sec23, which inhibits the budding of transport vesicles
off the ER membrane (resulting in retention of proteins in the
ER)(18, 23) . Strain RSY427 (sec23) (complete
genotypes of strains used in this study are described under
``Experimental Procedures'') containing expression plasmid
pA189invHD (15) was grown at 23 °C to log phase and then
preincubated for 1 h at 32 °C, a nonpermissive temperature for
strains containing sec23 and/or sec11 mutations (Fig. 2). Cells were pulse-labeled for 5 min with a mixture of
radiolabeled methionine and cysteine and then subjected to a chase for
90 min in the presence of excess unlabeled methionine and cysteine (see
``Experimental Procedures''). Proteins were precipitated from
cell extracts with anti-HD antibodies following the pulse and at
specific intervals during the chase. Immunoprecipitated proteins were
then resolved by SDS-PAGE and analyzed by fluorography. As shown in Fig. 3A, A189invHD was detected after the pulse in
strain RSY427 (SEC11 sec23) (lane1) and
then rapidly degraded during the 30- (lane2), 60- (lane3), and 90-min (lane4) chase
periods. The fact that A189invHD was degraded in the sec23 mutant, similar to that seen in wild-type cells(15) ,
demonstrated that A189invHD was degraded prior to its insertion into
transport vesicles that bud off the ER membrane. To determine the
effect of the sec11 mutation on A189invHD degradation, strain
CMY10 (sec11 sec23)/pA189invHD was examined by pulse-chase at
32 °C. This analysis revealed that A189invHD was only partially
degraded during a 90-min chase period in the sec11 sec23 double mutant (lanes5-8).
Figure 2:
Comparative growth analysis of wild-type, sec23 mutant, and sec11 mutant strains. Wild-type
strain FC2-12B (designated Sec
Figure 3:
Degradation of chimera A189invHD. A, strains RSY427 (SEC11 sec23) (lanes1-4) and CMY10 (sec11 sec23) (lanes5-8), each bearing pA189invHD, were subjected to a
pulse-chase analysis following a 1-h preincubation at a nonpermissive
temperature for both strains (32 °C). Labeled proteins were
precipitated with anti-HD antibodies after the 5-min pulse (P)
and at the indicated time points during the chase (C) period.
The position of A189invHD is indicated. B, quantitation of the
degradation of A189invHD in RSY427 (SEC11 sec23) (opensquares) and CMY10 (sec11 sec23) (closedsquares) was performed through scanning laser
densitometry on three independent analyses performed identically to
that depicted in A. Average values are plotted as percent of
A189invHD remaining relative to that in the pulse (100%) over time (in
minutes) of the chase.
Strain PBY408A (sec11) containing the plasmid bearing this new construct
(pAHDK2) was grown to log phase at 23 °C, followed by a
preincubation of 1 h at a nonpermissive temperature (37 °C). Cells
were then subjected to a pulse-chase analysis as described above,
except that proteins were precipitated from cell extracts with
anti-Kar2p antibodies. The temperature-induced defect in signal
peptidase activity in the sec11 mutant was readily apparent by
the presence of preKar2p throughout the pulse-chase analysis (Fig. 4, lanes5-8). At this
nonpermissive temperature, the chimera was stable during the 5-min
pulse (lane5) and the 90 min chase (lanes6-8). A plasmid containing the wild-type SEC11 gene (pCM111) was introduced into strain PBY408A (sec11)/pAHDK2 to further assess the role of signal peptidase.
Cells of this strain were examined by pulse-chase at 37 °C again
using anti-Kar2p antibodies. Evidence that pCM111 bearing SEC11 complemented the sec11 mutant was demonstrated by the
presence of mature Kar2p (lanes1-4), which has
an increased electrophoretic mobility on sizing gels as compared with
preKar2p. Importantly, the data show that upon restoration of signal
peptidase activity, a proteolytic fragment (f1) was produced during the
chase period (lanes2-4).
Figure 4:
Proteolysis of chimera AHDK2 in a sec11 mutant preincubated at the nonpermissive and
semipermissive temperatures. Strains PBY408A (sec11)/pAHDK2
(denoted as sec11) and PBY408A (sec11)/pAHDK2/pCM111
(containing SEC11) (denoted as sec11/SEC11) were
subjected to pulse-chase analyses following a 1 h preincubation at the
nonpermissive (37 °C) (lanes1-8) and
semipermissive (30 °C) (lanes9-15)
temperatures for the sec11 mutant. Labeled proteins were
precipitated with anti-Kar2p antibodies after the 5-min pulses (P) and at the indicated time points during the chase (C) periods. Relative positions of AHDK2, proteolytic
fragments f1 and f2 (indicated by the arrow), preKar2p (Kar2p
with signal peptide attached), and mature Kar2p (Kar2p with signal
peptide removed) are indicated.
The results described thus
far imply that signal peptidase was required for the cleavage of AHDK2
at two internal sites. The data did not, however, eliminate the
possibility that signal peptidase was indirectly responsible for the
proteolysis. Indeed, we reasoned that a 1-h preincubation at 30 and 37
°C prior to the pulse-chase analysis may have resulted in the
inactivation of a second protease dependent on signal peptidase for
enzymatic activity. In order for this scenerio to be plausible, most or
all of the putative second protease that was present in cells prior to
the 1-h preincubation must also have been inactivated. To examine the
effect of decreasing the preincubation period, we placed log phase
cells of strain PBY408A (sec11)/pAHDK2 at 30 °C for only 5
min before analysis by pulse-chase. The results in Fig. 5A, lanes1-3,
show that AHDK2 was stable for at least a 60-min chase period after the
5-min shift to 30 °C, whereas preKar2p was slowly converted to
Kar2p during the chase. Thus, in order for a second protease dependent
on signal peptidase for activity to be responsible for proteolysis of
AHDK2 a significant amount of the second protease present in cells
before the temperature shift would have to be inactivated during the
5-min preincubation. Although the data did not eliminate this
possibility, they did demonstrate that a small perturbation of Sec11p
more dramatically inhibits the proteolysis of AHDK2 than the cleavage
of the signal peptide of preKar2p.
Figure 5:
Pulse-chase and Western blot analyses of
AHDK2 proteolysis in a sec11 mutant under more permissive
conditions. A, strain PBY408A (sec11)/pAHDK2 was
subjected to a pulse-chase analysis after a 5-min preincubation at 30
°C. Labeled proteins were precipitated with anti-Kar2p antibodies
after the 5-min pulse (P) and at the indicated time points
during the chase (C). B, strain PBY408A (sec11)/pAHDK2 was analyzed by pulse-chase after a 1-h
preincubation at 28 °C. Labeled proteins were precipitated with
anti-Kar2p antibodies as in A. C, protein extracts
were prepared from strains FC2-12B (SEC11)/pAHDK2 (lanes3 and 5), PBY408A (sec11)/pAHDK2 (lanes2 and 4), and
control PBY408A (sec11) (lane1) grown at 19
°C and 23 °C. Extracts were resolved by SDS-PAGE and subjected
to Western blot analysis using anti-Kar2p antibodies. Positions of
several putative proteolytic fragments not seen in pulse-chase analyses
are indicated with arrows.
Figure 6:
Identification of the site of AHDK2
proteolysis. A, spheroplasts of wild-type strain FC2-12B
carrying pAHDK2 were homogenized and subjected to proteinase K
digestion for the indicated times with (lanes8-13) or without (lanes2-7) detergent. Control lanes2 and 8 represent homogenates not receiving proteinase K.
Processing of all samples required approximately 15 s and is calculated
into the time designations. Whole cell extract from strain
FC2-12B/pAHDK2 (designated Ctl) is represented in lane1 and is present to indicate the relative
positions of AHDK2, f1, f2, and Kar2p. The position of a proteolytic
fragment (Kar2f) of endogenous Kar2p is indicated. Samples were
resolved by SDS-PAGE and subjected to Western blot analysis using
anti-Kar2p antibodies. B, strains CSa42 (sec61)/pAHDK2 (lanes1-2) and RSY427 (sec23)/pAHDK2 (lanes3-4) were
subjected to a pulse-chase analysis following a 1-h preincubation at a
nonpermissive temperature (37 °C). Proteins were precipitated with
anti-Kar2p antibodies after the pulse (P) and at 45 min into
the chase periods (C). As demonstrated in Fig. 7, AHDK2
in the sec61 mutant is not glycosylated and thus has a higher
electrophoretic mobility on sizing gels. These data show a small amount
of preKar2p after the 5-min pulse (lane3), probably
because of a general defect in protein trafficking, due to the presence
of the sec23 mutation in
cells(23) .
Figure 7:
Assay for membrane integration and
glycosylation of AHDK2. Strains CSa42 (sec61)/pAHDK2 (lanes1 and 3), FC2-12B (SEC11)/pAHDK2 (designated Sec
Figure 8:
Pulse-chase analysis of AHDK2 proteolysis
using anti-Kar2p and anti-HD antibodies. Cells of wild-type strain
FC2-12B (SEC11)/pAHDK2 were subjected to pulse-chase
analyses at 30 °C using anti-Kar2p antibodies (lanes1-3) or anti-HD antibodies (lanes4-5). Labeled proteins were precipitated after the
5-min pulse (P) and at the indicated time points during the
chase (C).
Figure 9:
Cellular fractionation of strain FC212-B (SEC11)/pAHDK2. Whole cell extracts of wild-type strain
FC2-12B(SEC11)/pAHDK2 were prepared and fractionated as
described under ``Experimental Procedures.'' Lanes1-3 represent whole cell extract, high speed
supernatant fraction, and high speed pellet fraction respectively.
Samples were resolved by SDS-PAGE and subjected to Western blot
analysis using anti-Kar2p antibodies. Relative positions of AHDK2, f1,
f2, and endogenous Kar2p are indicated. A putative proteolytic fragment
of AHDK2 is indicated by the arrow.
Chimeric membrane protein A189invHD appears to be degraded by
at least two pathways within the lumen of the ER: one greatly
facilitated by signal peptidase (specifically the Sec11p subunit) and a
second independent of signal peptidase. As shown in Fig. 3, A and B, A189invHD is degraded by more than 95% in
cells expressing functional signal peptidase, while degradation is
significantly reduced in cells containing a conditional defect in
Sec11p (approximately 60% degraded). The 40% of A189invHD apparently
requiring signal peptidase for its degradation may be resistant to the
signal peptidase-independent pathway due to the adoption of an
insensitive conformation or posttranslational modifications at
proteolytically susceptible sites. In this regard, we have previously
shown that A189invHD is glycosylated prior to its complete degradation (15) . An alternative explanation for the fraction of
relatively stable A189invHD in the sec11 mutant is that a
fraction of the chimera may be transported away from the ER degradation
machinery (i.e. to a different cellular compartment). However,
through the use of the sec23 sec11 double mutant, any
trafficking of the chimera into the Golgi apparatus is probably
eliminated.
While A189invHD is apparently susceptible to two
proteolytic pathways in the ER, at least one of which involves the
endoprotease signal peptidase, no proteolytic fragments are observed.
In an attempt to identify and analyze proteolytic intermediates, we
constructed AHDK2, a derivative of A189invHD in which a portion of the
carboxyl-terminal HD sequence was replaced by Kar2p, a resident protein
of the ER lumen (Fig. 1). Upon examination of AHDK2 in cells
expressing signal peptidase, we observed that the chimera is processed
within the ER with the production of two proteolytic fragments (f1 and
f2) (Fig. 4, 6, and 8). Both fragments contain all or part of
the carboxyl-terminal Kar2p sequence as demonstrated by
immunoprecipitation studies with carboxyl-terminal anti-Kar2p
antibodies (Fig. 8). In contrast, AHDK2 is stable in the sec11 mutant at the nonpermissive and semipermissive
temperatures, whereas preincubation of cells at a more permissive
temperature results in slight but detectable degradation. These data
implicate signal peptidase directly in the proteolytic event and
provide the strongest evidence to date that signal peptidase can cleave
internal sites of polypeptide chains.
Though f1 and f2 appear
relatively stable in our pulse-chase analyses, Fig. 4and Fig. 8show that f1 is slowly degraded while f2 slowly
accumulates in wild-type cells and in the sec11 mutant
complemented with SEC11. Because these fragments contain the
Kar2p sequence, they may acquire protection normally afforded to ER
resident proteins. Such naturally conferred protection could result
from a variety of means, including the adoption of a tertiary
structure, which sequesters potential cleavage sites, and associations
with other proteins within the ER, again hiding sensitive sequences.
The presence of the carboxyl-terminal Kar2 sequence not only appears to
have stabilized two proteolytic intermediates but also may have
prevented degradation of the intact chimera by the signal peptidase
(Sec11p)-independent pathway since, unlike A189invHD, AHDK2 is stable
in a sec11 mutant at the nonpermissive temperature.
Since
signal peptide cleavage sites contain small uncharged or neutral
residues at the -1 position preceded by a series of
hydrophophobic amino acids(37) , the proximity of the f1
cleavage site to the transmembrane segment suggests that the
hydrophobic residues of the transmembrane segment of AHDK2 may be
important for targeting the f1 cleavage site to signal peptidase. In
this regard, the transmembrane segment derived from arginine permease (15, 17) contains, at the carboxyl-terminal end,
alanine and serine residues that are commonly found at the -1
position from signal peptide cleavage sites. The f2 cleavage site of
AHDK2, however, is located in the carboxyl-terminal part of the HD
moiety (derived from a cytoplasmic enzyme), which is separated from the
transmembrane segment by more than 300 amino acids. Interestingly, this
region of HD contains two stretches of uncharged amino acids (residues
480-507 and residues 539-555) that are followed by
alanine(7) . If one or both of these stretches functions in the
targeting of the f2 site to signal peptidase, then it is plausible that
uncharged stretches of amino acids within other abnormal ER proteins
serve a similar function.
It has previously been shown that signal
peptides are cleaved in a cotranslational manner(3) . Since
numerous proteins targeted for ER degradation are susceptible after a
postinsertion lag period (for review, see Ref.19), this suggests that
for signal peptidase to play a general role in ER degradation, its
recognition of specific cleavage sites may not necessarily occur
cotranslationally. There are two instances in our study in which
post-translational cleavage events presumably mediated by signal
peptidase are detected in wild-type and mutant cells: AHDK2 is cleaved
in a signal peptidase-dependent manner after it is fully synthesized
and glycosylated and preKar2p is converted to Kar2p after a 30-min
chase period in the sec11 mutant at the semipermissive
temperature. These results therefore show that the active site of
signal peptidase is accessible to polypeptides after they have been
translocated to the ER lumen.
We thank those who provided antibodies, strains, and
plasmids.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)membrane, their
amino-terminal signal peptides are removed by signal peptidase through
an endoproteolytic cleavage reaction at the luminal side of the
membrane(3, 32, 35) . This proteolytic
process is one of a series of events required for the maturation of
newly synthesized proteins within the ER, including asparagine-linked
core glycosylation, disulfide bond formation, and the assembly of
multisubunit complexes (for review, see (11) and (24) ). In vivo studies in yeast show that ablation of
the maturation process as a result of a temperature-sensitive mutation
affecting signal peptidase activity (sec11) leads to a
deficit in protein trafficking to the cell surface and lethality at the
nonpermissive temperature(4) .
Media, Strains, and Antibodies
YPD (rich),
SC-CAS (supplemented), and SC (minimal) media have been described
previously (15) . SC-CAS medium contains leucine and histidine
as part of a casamino acids supplement but is deficient in uracil,
adenine, and tryptophan. Nucleotides and amino acids were added when
appropriate to a final concentration of 0.1 mg/ml each. When selecting
for plasmids containing URA3, SC-CAS supplemented with
tryptophan was used. SC medium, with appropritate supplements for
auxotrophs, was used for growth of cells containing plasmids bearing LEU2. The yeast strains used in this work are as follows:
FC2-12B, MAT trp1-1 ura3-52 his4-401
leu2-1 HOL1-1 CAN1-1(15) ; CSa42, MATasec61-3 trp1-1
ura3-52 his4-401 HOL1-1(13) ; RSY427, MAT
sec23-1 ura3-52 leu2-1 trp1-1
his4-619 (from Jon Rothblatt, Dartmouth College, Hanover,
NH); PBY408A, MAT
sec11-7 ura3-52 his4-519
leu2-3,112 gal2-1(4); and CMY10, MAT
sec23-1 sec11-7 trp1-1 ura3-52 his4 leu2 (constructed in this study from strains PBY408A and RSY427).
Anti-Kar2p was from Mark Rose (Princeton University);
anti-carboxypeptidase Y was from Randy Schekman (University of
California, Berkeley); and anti-invertase was from Johnny Ngsee
(Stanford University, Stanford, CA). Anti-HD antibodies were described
previously(15) .
Plasmid Constructions
Construction of pA189invHD
(2 µ URA3) has been described(15) . pAHDK2 (2
µ URA3) was made as follows. The PvuII
restriction fragment was removed from plasmid pA189invHD, and a NotI linker (GCGGCCGC) was inserted into this PvuII
site. A fragment of the KAR2 gene encoding yeast BiP (22, 25) was amplified from pMR713 (CEN4
LEU2) (from Dr. Mark Rose) by the polymerase chain reaction using
oligonucleotide primers (AAGCGGCCGCCCTTTACAGAATTCTTTCCA and
TTGCGGCCGCAATTGTATGAAGCTCGAAGT), corresponding to sequences near KAR2 (nucleotides 81-100, where nucleotide 1 is the
beginning of the protein coding sequence, and nucleotides
2156-2175, which extend beyond the protein coding sequence). Each
primer contains a NotI restriction site. The amplified DNA
fragment was restricted with NotI and inserted into pA189invHD
containing the above-mentioned NotI linker. The orientation of
the inserted fragment was determined by restriction analysis, revealing
pAHDK2, which encodes an in-frame fusion between HD and Kar2p (see Fig. 1). The sequence of the HD-Kar2 fusion joint is
SIDMPGGRPLQNSF, where DMP is the end of the HD fragment and PLQ is the
beginning of the Kar2 fragment. For the construction of pCM111 (CEN6 LEU2), a 1.3-kilobase SacI-BamHI
restriction fragment (containing the wild-type SEC11 gene with
its endogenous promoter) was isolated from plasmid pRSB224 (2 µ URA3) (a gift of Dr. Randy Schekman). This fragment was
subsequently inserted into the polylinker region of pRS315 (CEN6
LEU2)(28) .
Pulse-Labeling, Pulse-Chase Analyses, Western Blotting,
and Quantitation of Protein Levels
Temperature-sensitive strains
(PBY408A, CSa42, RSY427, and CMY10) were grown at the permissive
temperature (23 °C) to A = 1 (log
phase) in appropriate media for selection of plasmids (above) and then
shifted to SC medium, with appropriate supplements but lacking
methionine and cysteine, for 1 h at 23 °C and then to 28 °C for
1 h, 30 °C for 5 min, 30 °C for 1 h, 32 °C for 1 h, or 37
°C for 1 h before the addition of label. Nonconditional strain
FC2-12B bearing one of the above-mentioned plasmids containing URA3 was grown at 30 °C to A
= 1 in SC-CAS medium, supplemented appropriately, and then
shifted to methionine- and cysteine-depleted medium (above) for 1 h
before the addition of label. Cells were subjected to a 5-min pulse
with 16 µCi/ml of a protein labeling mix containing
[
S]methionine and
[
S]cysteine (EXPRE
S
S,
DuPont NEN) and in some experiments to a chase with excess unlabeled
methionine and cysteine (added to a final concentration of 800
µg/ml methionine and 200 µg/ml cysteine). Cells were removed at
the time intervals indicated in the text. In all experiments, an equal
amount of cells was removed at each interval (1 A
equivalent of cells).
cells), or anti-HD (3 µl/1 A
cells) to the supernatant followed by an
overnight incubation on ice. The following day a mixture containing
phosphate-buffered saline, 1% Triton X-100, protein A-insoluble
lyophilized cell powder was prepared by the instructions provided by
the supplier (Sigma). A fraction of the mixture (10 µl) was added
to each Eppendorf tube, and the tubes were subsequently placed on a
rotating platform for 45 min. The cell powder containing bound
antibodies was sedimented for 30 s in a microcentrifuge (12,000 rpm),
and the pellet was subjected to two washes with phosphate-buffered
saline, 1% Triton X-100 followed by two washes with distilled water.
The pellet was boiled in SDS-PAGE sample buffer (20 µl) for 5 min
and subjected to SDS-PAGE on a 7% polyacrylamide gel. Following
electrophoresis, gels were stained with Coomassie Blue, destained,
washed in water, and then incubated in a solution containing sodium
salicylate (1 M) for 30 min. The gel was then dried and placed
on x-ray film for analysis by fluorography.
Cellular Fractionation, Spheroplast Preparation, and
Proteinase K Treatment of Cell Extracts
For cellular
fractionation studies, cells of wild-type strain
FC2-12B(SEC11)/pAHDK2 (3 mls) were grown to A = 1 (log phase) in appropriate media
for selection of plasmid. Cellular extract from 1 OD equivalent (1 ml)
was prepared as described above and resuspended in SDS-PAGE sample
buffer (10 µl). Remaining 2 OD equivalents (2 mls) were pelleted
and resuspended in 400 ml of fractionation buffer containing protease
inhibitors(15) . The cells were then lysed using glass beads.
This suspension was then subjected to a low speed spin (10,000
g for 5 min) at 4 °C. The supernatant (
400 µl)
was removed and subjected to a high speed spin (45,000
g for 45 min) at 4 °C. Resulting high speed supernatant
(representing soluble fraction) was removed and added to 400 µl of
ice cold 20% trichloroacetic acid. Proteins were precipitated as above
and resuspended in 10 µl of SDS-PAGE sample buffer. The high speed
pellet (representing membrane fraction) was directly dissolved in 10
µl of SDS-PAGE sample buffer. Whole cell protein extract, soluble
fraction, and membrane fraction were boiled and resolved on SDS-PAGE
followed by Western blot analysis using anti-Kar2p antibodies (25) (1:5000 dilution).
= 1 (log phase) in appropriate media for selection of
plasmid. Spheroplasts preparation and lysis were then performed at 4
°C as described previously(8) . Spheroplast homogenate was
subjected to a low speed spin (650
g for 4 min) at 4
°C to pellet unbroken cells. Resulting supernatant was then split
into two equal volumes (approximately 6 OD equivalents/tube). One tube
was adjusted to 0.4% Triton X-100 with the second designated detergent
free. A volume representing 1 OD equivalent was immediately removed
from each tube and quenched in a 14
volume of 20%
trichloroacetic acid. Proteinase K (U. S. Biochemical Corp.) was then
added to remaining 5 OD equivalents in each tube to a final
concentration of 0.3 mg/ml. A volume representing 1 OD equivalent was
immediately removed from each tube and quenched as above. Sample
collection of these and following fractions required approximately 15
s. At subsequent time points indicated in the text, volumes
representing 1 OD equivalent were removed from each tube and quenched
in trichloroacetic acid. Proteins were precipitated by centrifugation
(12,000 rpm/5 min) in a tabletop microcentrifuge. Protein pellets were
resuspended in 10 µl of SDS-PAGE sample buffer. Boiled samples were
resolved by SDS-PAGE and subjected to Western blot analysis using
anti-Kar2p antibodies (1:5000 dilution).
The Degradation of A189invHD Is Facilitated by Signal
Peptidase
We previously constructed a series of gene fusions for
the purpose of identifying topologically distinct domains of arginine
permease, a multispanning membrane protein of the yeast S.
cerevisiae(15) . Pulse-chase analyses revealed that some
of the chimeras were rapidly degraded in vivo. One such
chimera, A189invHD, was comprised of 189 amino-terminal residues of
arginine permease (including a type II transmembrane segment), a spacer
sequence derived from invertase, and the cytoplasmic enzyme histidinol
dehydrogenase (HD) (Fig. 1). Topology studies indicated that
100 residues of the amino terminus of A189invHD were placed on the
cytoplasmic side of the membrane followed by a signal/anchor sequence
(transmembrane segment) and then by the carboxyl-terminal
invertase-histidinol dehydrogenase sequence(14, 15) .
Integration of A189invHD into the ER membrane resulted in the
translocation of the invertase-histidinol dehydrogenase moiety to the
lumen, where glycosylation at one or more of the 11 glycosylation sites
in the invertase-histidinol dehydrogenase sequence
occurred(15) .
) and
conditional mutant strains RSY427 (sec23) and PBY408A (sec11) were placed on agar plates containing YPD (rich)
medium and then incubated at 23, 30, and 32 °C for 3-5 days.
Diagram in lowerright depicts the arrangement of the
strains under study.
Quantitation
of A189invHD degradation kinetics revealed that >95% of A189invHD
was degraded during the first 30 min of the chase in strain RSY427 (SEC11 sec23), while only 60% of A189invHD was rapidly
degraded in strain CMY10 (sec11 sec23) (Fig. 3B). These data suggest that the degradation of
approximately 40% of A189invHD required expression of functional signal
peptidase (specifically Sec11p), while a significant fraction (60%) was
proteolyzed through a signal peptidase-independent process. Thus,
despite the absence of proteolytic intermediates, these data clearly
implicated signal peptidase (an endoprotease) in the degradation of
A189invHD.
Fusion of Kar2p to the Carboxyl Terminus of A189invHD
Leads to the Production of Relatively Stable Proteolytic
Intermediates
To further investigate the role of signal
peptidase, we constructed a second gene fusion termed AHDK2 (see
``Experimental Procedures''). AHDK2 differs from its
progenitor, A189invHD, in its carboxyl terminus at which 207
carboxyl-terminal residues of HD were replaced with residues
28-682 of the ER-resident protein Kar2p (Fig. 1). Residues
28-682 represent all of the sequences of full-length Kar2p
lacking the positively charged region and the hydrophobic core sequence
of its amino-terminal signal peptide(22) . The Kar2p sequence
was utilized for the following reasons. First, Kar2p, a resident
luminal protein presumably not targeted for ER degradation, may
stabilize all or part of the sequences to which it is attached, thereby
leading to proteolytic intermediates. Second, antibodies directed
against Kar2p can be used in conjunction with anti-HD antibodies to
determine the fate of different sequences within the polypeptide chain.
Third, endogenous Kar2p provides an internal control for all analyses
performed with anti-Kar2p antibodies.
To decrease the
likelihood that high temperature-induced cellular aberrations unrelated
to Sec11p are the cause for the chimera's stability in the sec11 mutant, we also performed the above analysis at 30
°C, a semipermissive (nonlethal) temperature for the sec11 mutant (the growth pattern of the sec11 mutant is shown
in Fig. 2). Log-phase cells of strain PBY408A (sec11)/pAHDK2 bearing pCM111 (SEC11) or not bearing
pCM111 were preincubated at 30 °C for 1 h and then subjected to a
pulse-chase analysis. That the semipermissive temperature inhibited
signal peptidase activity was demonstrated by the presence of preKar2p
in the pulse (Fig. 4, lane13). Interestingly,
preKar2p was slowly converted to mature Kar2p over the 60-min chase
period (lanes14 and 15), suggesting the
signal peptide of preKar2p could be cleaved in a post-translocational
manner. Incubation of the sec11 mutant at the semipermissive
temperature, however, resulted in strong inhibition in degradation of
AHDK2 (compare the 5-min pulse, lane13, to the 30-
and 60-min chase periods, lanes14and 15). Upon restoration of signal peptidase activity (by the
introduction of pCM111 into the sec11 mutant), the chimera
suffered proteolysis with the production of f1 and a second proteolytic
fragment (f2) (lanes9-12), which was derived
from f1 (note that f1 and f2 were precipitated with antibodies directed
against the carboxyl terminus of Kar2p). The results shown in Fig. 4demonstrated that, unlike A189invHD, which was only
partially stable in the sec11 mutant, AHDK2 was resistant to
proteolysis in the sec11 mutant. Furthermore, the fusion of
Kar2p to the carboxyl terminus of A189invHD (thus producing AHDK2)
resulted in two proteolytic fragments.
A criterion that can be used to
assess whether a temperature-sensitive mutation directly inhibits the
process in question is to show temperature dependence. Since AHDK2 was
stable in the sec11 mutant at 30 and 37 °C, its
degradation was next analyzed at more permissive temperatures. Strain
PBY408A (sec11)/pAHDK2 was grown to log-phase at 23 °C,
shifted to 28 °C for 1 h, and then analyzed by pulse-chase. Signal
peptidase was only partially inactivated under these conditions as
precursor and processed forms of Kar2p were present after the 60-min
chase period (Fig. 5B, lane3).
Similarly, partial degradation of AHDK2 was observed at 28 °C
(compare lanes1 and 3). The data depicted
in Fig. 4and Fig. 5thus demonstrate that AHDK2 was
proteolyzed in the sec11 mutant in a temperature-dependent
manner; however, f1 and f2 were not detected in the sec11 mutant at 28 °C. Reasoning that proteolytic fragments may be
produced at a slow rate in the sec11 mutant, strain
PBY408A(sec11)/pAHDK2 and control strain
FC2-12B(SEC11)/pAHDK2 were grown to log phase at 19 and
23 °C and then analyzed by steady-state methods. The Western blot
depicted in Fig. 5C revealed that AHDK2, f1, and f2
were present in strain FC2-12B(SEC11)/pAHDK2 at 19 and
23 °C (lanes3 and 5). The sec11 mutant carrying pAHDK2 (lanes2 and 4)
contained AHDK2 and f2, but not f1, presumably because cleavage at the
f1 site of chimera AHDK2 infrequently occurs relative to other
cleavages of AHDK2 in sec11 mutant cells at permissive
temperatures. Of course, if the f2 cleavage site or another cleavage
site was recognized first, f1 would be eliminated apriori. As expected, we did not detect AHDK2 or
proteolytic fragments in control strain PBY408A (sec11)
lacking the expression plasmid (lane1). By comparing
this control to lanes corresponding to the sec11 mutant (lanes2 and 4) and wild-type strain
FC2-12B bearing pAHDK2 (lanes3 and 5), it appears that additional fragments of AHDK2 were present
in cells analyzed in the steady state. These fragments were not
detected in the pulse-chase analysis and therefore may accumulate
slowly in cells. In sum, these data support the idea that signal
peptidase is directly responsible for the cleavage of AHDK2 at one or
more internal sites.
Proteolysis of AHDK2 Occurs within the ER
Lumen
The apparent involvement of signal peptidase in the
proteolysis of AHDK2 suggested that f1 and f2 may be produced within
the ER. Using an in vitro approach, we asked whether f1 and f2
were contained within a membrane compartment that could afford
protection from externally added proteases. Spheroplasts were prepared
from log phase cells of strain FC2-12B/pAHDK2, lysed under
conditions that resulted in minimal release of luminal proteins,
followed by treatment of cellular homogenates with proteinase K (see
``Experimental Procedures''). As shown in Fig. 6A, f1 and f2 were protected from proteinase K in
membrane preparations lacking detergent (lanes3-7). Upon solubilization of the membranes through
the addition of the detergent Triton X-100 (lanes9-13), f1 and f2 were degraded by proteinase K.
Most of Kar2p, a ER luminal protein, was also protected in preparations
lacking detergent (lanes3-7); however, a
fraction of Kar2p was cleaved producing a smaller molecular weight
fragment (Kar2f). This fragment probably represented Kar2p that was
released from membrane vesicles during their preparation since all of
Kar2p was converted to Kar2f upon the addition of proteinase K to
detergent-solubilized membranes (lanes9-13).
This biochemical analysis demonstrated that f1, f2, and, as expected,
Kar2p were sequestered within a membrane compartment. Surprisingly,
intact AHDK2 was not detected in membrane preparations lacking
proteinase K (lanes2 and 8), while it was
present in extracts prepared by breaking cells in a solution of 10%
trichloroacetic acid to inactivate endogenous proteases (lane1). The absence of AHDK2 may have been due to signal
peptidase which, of course, would be present in the ER membrane and
remain active during the preparation of microsomal membranes.
) (lane2), and PBY408A (sec11)/pAHDK2 (lane4) were subjected to a 5-min pulse analysis
following a 1-h preincubation of cells at 37 °C (a nonpermissive
temperature for the sec61 and sec11 mutants)(4, 30) . Labeled proteins were
precipitated with anti-HD antibodies (lanes1-4). The relative position of glycosylated AHDK2
is indicated, while the nonglycosylated form of AHDK2 is denoted by the asterisk.
A
genetic analysis was next performed to determine whether f1 and f2 were
produced specifically within the ER. Two conditional mutants, one
inhibiting membrane protein integration, CSa42 (sec61) (30) and a second inhibiting the budding of transport vesicles
off the ER membrane, RSY427 (sec23)(23) , were
transformed with pAHDK2 and then analyzed by pulse-chase at the
nonpermissive temperature (37 °C). When protein integration into
the membrane was blocked in the sec61 mutant, degradation and
subsequent production of f1 and f2 did not occur during the 5-min pulse
or 45-min chase (Fig. 6B, lanes1 and 2). Translocation of endogenous Kar2p was blocked in the sec61 mutant as demonstrated by the presence of preKar2p (lanes1 and 2). However, upon examination
of the sec23 mutant, the two diagnostic fragments were present (lanes3and 4). The sec23 mutation inhibited protein transport to the Golgi apparatus since
a control analysis demonstrated that the core-glycosylated precursor to
vacuolar carboxypeptidase Y(29) , was not converted to the
mature form in strain RSY427 (sec23) at 32 or 37 °C (data
not shown). Taken together, the data depicted in Fig. 6demonstrated that AHDK2 was proteolyzed within the ER.
Integration and Glycosylation of AHDK2 in the sec11
Mutant
Since the results in Fig. 6B revealed
that AHDK2 was stable in the sec61 mutant, we next determined
whether the stability of AHDK2 in the sec11 mutant could be
attributed to a defect in integration. To monitor integration, we
assayed for the increase in molecular weight associated with
glycosylation of the invertase-histidinol dehydrogenase sequence, which
contains eight potential glycosylation sites (the arginine permease and
Kar2p sequences do not contain core-glycosylation sites) (see Fig. 1for a description of AHDK2). The expression plasmid pAHDK2
was introduced into strains CSa42 (sec61), PBY408A (sec11), and control strain FC2-12B (SEC11).
Log-phase cells were shifted to the nonpermissive temperature for the sec61 and sec11 mutants (37 °C) for 1 h and then
pulse-labeled for 5 min with radiolabeled methionine. Proteins were
precipitated from cell extracts with anti-HD antibodies and analyzed by
SDS-PAGE and fluorography. AHDK2 from the sec61 mutant (Fig. 7, lanes1and 3)
exhibited an increase in mobility on sizing gels compared with that
from wild-type (lane2) and sec11 cells (lane4), indicating that the sec61, but not
the sec11, mutation inhibited integration and subsequent
glycosylation of AHDK2. This is consistent with in vitro studies in mammalian systems demonstrating that the SPC is not
required for the insertion of type I and type II membrane proteins into
reconstituted membrane vesicles(12) . In addition, Fig. 7shows that AHDK2 was glycosylated before significant
levels of proteolysis occurred.
Positioning of the f1 and f2 Cleavage Sites
As
described above, the availability of various antibodies recognizing
AHDK2 can be used to assess the fate of distinct regions within the
chimera. Furthermore, the use of different antibodies should enable us
to localize the f1 and f2 cleavage sites to specific regions within the
polypeptide chain. Strain FC2-12B (SEC11) bearing the
expression plasmid pAHDK2 was subjected to pulse-chase analyses using
anti-Kar2p and anti-HD antibodies. As expected, f1 and f2 were
precipitated with anti-Kar2p antibodies (Fig. 8, lanes1-3). In contrast, f1, but not f2, was precipitated
with anti-HD antibodies (lanes4-5), indicating
that the sequence comprising the anti-HD recognition site (see legend
to Fig. 1) was absent from f2. Based on the sizes of f2 (90
kDa) and Kar2p (79 kDa) and the fact that the anti-HD antibodies are
directed against the amino-terminal part of the HD moiety, we conclude
that the f2 cleavage site is located within the carboxyl-terminal
region of HD.
As other type II membrane proteins are cleaved near
their transmembrane segments, putatively by signal peptidase, it seemed
plausible that the f1 cleavage site was near the transmembrane segment
of AHDK2. If correct, this suggested that f1 (and the smaller fragment
f2) may be liberated from the ER membrane. To address this, log-phase
cells of strain FC2-12B(SEC11)/pAHDK2 were broken by
vortex mixing with glass beads in a buffer containing a
protease-inhibitor mixture (see ``Experimental Procedures'').
Cell extracts were then fractionated by differential centrifugation and
subjected to Western blot analysis with anti-Kar2p antibodies. As
expected, AHDK2 was located exclusively in the membrane pellet (Fig. 9, lane3). The majority of f2 was
located in the supernatant fraction (lane2). In
contrast to the clean separation of apparently soluble f2 and
membrane-bound AHDK2, f1 fractionated in both the membrane pellet and
the supernatant fraction. This indicated that a significant amount of
f1 was released from the membrane, thus suggesting that the f1 cleavage
site was located at the luminal side of the transmembrane segment. In
addition, we have shown that f1 but not f2, is recognized by antibodies
directed against invertase (data not shown), further localizing the f1
cleavage site between the transmembrane segment and the
carboxyl-terminal part of the invertase moiety (see Fig. 1). Fig. 9also revealed that approximately 50% of endogenous Kar2p,
a protein lacking an apparent transmembrane segment, was present in the
membrane pellet. This finding is consistent with the idea that Kar2p
may interact with Sec63p, a ER membrane protein(6) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.