©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mutation Affecting Signal Peptidase Inhibits Degradation of an Abnormal Membrane Protein in Saccharomyces cerevisiae(*)

(Received for publication, April 14, 1995)

Chris Mullins (§) , YiQi Lu , Allyson Campbell (¶) , Hong Fang , Neil Green

From the Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232-2363

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

During or immediately following the translocation of pre-proteins across the ER()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) .

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.


EXPERIMENTAL PROCEDURES

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) .


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.



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 (EXPRESS, 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).

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 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.

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.

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).

For proteinase K protection analysis, cells of wild-type strain FC2-12B (SEC11)/pAHDK2 (12 mls) were grown to A = 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).


RESULTS

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) .

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) 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.




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.



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.

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.



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.

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.



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.


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) (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.


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).



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) .


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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by the Department of Health and Human Services Training Grant 2 T32 CA09385-11 (to C. M.), by the American Cancer Society Institutional Research Grant IN-25-32, and the National Institutes of Health Biomedical Research Support Grant RR-05424 (to H. F.), and by grants from the American Cancer Society and the Joe C. Davis Foundation (to N. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 615-343-0453; Fax: 615-343-7392.

Present address: Dept. of Molecular Physiology and Biophysics, Vanderbilt Medical School, Nashville, TN 37232-2363.

The abbreviations used are: ER, endoplasmic reticulum; SPC, signal peptidase complex; PAGE, polyacrylamide gel electrophoresis; OD, optical density; HD, histidinol dehydrogenase.


ACKNOWLEDGEMENTS

We thank those who provided antibodies, strains, and plasmids.


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