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Originally published In Press as doi:10.1074/jbc.M703200200 on July 10, 2007

J. Biol. Chem., Vol. 282, Issue 37, 27402-27413, September 14, 2007
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The Proprotein Convertase SKI-1/S1P

ALTERNATE TRANSLATION AND SUBCELLULAR LOCALIZATION*

Philomena Pullikotil{ddagger}, Suzanne Benjannet{ddagger}, Janice Mayne§, and Nabil G. Seidah{ddagger}1

From the {ddagger}Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada and §Hormones, Growth, and Development, Ottawa Health Research Institute, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada

Received for publication, April 16, 2007 , and in revised form, July 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Subtilisin kexin isozyme-1 (SKI-1) represents the first mammalian member of secretory subtilisin-like processing enzymes that cleaves after nonbasic residues. It is synthesized as an inactive precursor that undergoes three sequential autocatalytic processing steps of its N-terminal prosegment and an ectodomain shedding at a site near the transmembrane domain. The various cellular functions of SKI-1 emphasize the need to understand the sites of its activation and shedding. We have previously shown that SKI-1 undergoes autocatalytic shedding at the sequence KHQKLL953{downarrow}, resulting in a membrane-bound stump called St-1 (amino acids 954-1052). However, little is known about the cellular localization of SKI-1 or its shed forms. In the present study, we have further identified a smaller C-terminal fragment St-2 generated closer to the transmembrane domain. By sequencing and mass spectrometric analysis, the start site and the molecular mass of St-2 were determined. Site-directed mutagenesis revealed the critical amino acid involved in this novel process. Mutation of Met990 to M990A, M990I, and M990L failed to generate St-2, suggesting an internal alternate translation event at Met990, as confirmed by an in vitro transcription/translation assay. Confocal microscopy defined the subcellular localization of SKI-1 and its fragments. The data show that most of membrane-bound SKI-1 and its stumps St-1 and St-2 localize to the Golgi and can enter the endosomal/lysosomal compartments but do not sort to the cell surface. Deletion studies showed that the transmembrane domain of SKI-1 determines its trafficking. Finally, rSt-1 and rSt-2 seem to affect the processing of ATF6 by SKI-1, but cellular stress does not regulate the production of St-2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several secretory proteins are synthesized as inactive precursors, which when converted to their mature forms by proteolytic enzymes generate a large diversity of bioactive proteins and peptides. The nine-member family of the proprotein convertases (PCs)2 participates actively in the generation of such molecular diversity (1-3). There are seven basic amino acid-specific kexin-like mammalian proprotein convertases that cleave various precursors at the general consensus motif (K/R)Xn(K/R){downarrow}, where n = 0, 2, 4, or 6, and X represents any amino acid. The eighth member is the pyrolysin-like subtilisin kexin isozyme-1 (SKI-1) (4), also known as site-1 protease (S1P) (5). It cleaves substrates at the consensus motif (R/K)X-(hydrophobic)-X{downarrow}, where X is variable (6). The last member PCSK9 cleaves the sequence VFAQ152 {downarrow} within its prosegment (7).

SKI-1 represents the first mammalian member of secretory subtilisin-like processing enzymes that cleaves after nonbasic residues (2, 3). The ubiquitously expressed convertase SKI-1 (4) regulates the synthesis of cholesterol and fatty acids and their metabolism, through the processing of the membrane-bound transcription factors sterol regulatory element-binding proteins (8, 9). It also regulates endoplasmic reticulum (ER)-stress response through cleavage of ATF6 (10) and is involved in the processing of probrain-derived neurotrophic factor (4). Recently, SKI-1 was reported to process Luman, a basic leucine zipper transcription factor, as well as other CREB-like bZIP factors (11). The enzyme also plays a major role in the processing of surface glycoproteins of infectious viruses, such as Lassa (12), lymphocytic choriomeningitis (13), and Crimean Congo hemorrhagic fever viruses (14).

SKI-1 is synthesized as an inactive precursor of 1052 amino acids (aa) that is activated following three sequential autocatalytic processing events within its prosegment (sites B/B' and C; Fig. 1A) (15). The membrane-bound SKI-1 is rendered soluble via an autocatalytic ectodomain shedding event at a KHQKLL953 {downarrow} site near the transmembrane domain (Fig. 1A) (15), similar to many cell surface proteins (e.g. growth factors, cytokine, growth factor receptors, and beta-amyloid precursor proteins) (16). Shedding of SKI-1 releases a soluble protein soluble SKI-1 (aa 188-953), leaving behind a membrane-bound stub (St-1; aa 954-1052) (Fig. 1A). The biological importance of the shedding and the role, if any, of the membrane-bound stub St-1 is unknown.

In the present study, we have identified a new mechanism generating a smaller membrane-bound C-terminal fragment, denoted as St-2 (Fig. 1A). Microsequencing and mass spectrometric data revealed that St-2 starts at Met990. Site-directed mutagenesis and in vitro transcription/translation indicated a unique alternate translation event starting at Met990. Immunocytochemistry performed on endogenous and overexpressed SKI-1 and various constructs, including those coding for St-1 and St-2, revealed that SKI-1 can sort to endosome/lysosomes but not to the cell surface. These stumps seem to interfere with the ability of SKI-1 to process pro-ATF6 into its nuclear form in response to cellular stress.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human embryonic kidney (HEK293) cells were grown in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum. Chinese hamster ovary (CHO)-K1, human hepatocytes (HuH7), M19 cells lacking site-2 protease S2P (17), and Neuro 2A were grown in Ham's Dulbecco's modified Eagle's medium/F-12 medium containing 10% fetal bovine serum. SRD-12B cells lacking SKI-1/S1P expression (18) were cultured in a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 µg/ml streptomycin sulfate supplemented with 5% fetal bovine serum, 5 µg/ml cholesterol, 1 mM sodium mevalonate, and 20 µM sodium oleate.

Recombinant Stump-1 (rSt-1) and Its Mutants—All oligonucleotides used in the various constructions are listed in Table 1. To obtain enough protein for sequencing and mass spectrometric analysis, the C-terminal fragment of human SKI-1 (aa 954-1052) rSt-1 was amplified by PCR using pIRES-SKI-1-V5 cDNA (4) as template, using primers S1/AS1 with XhoI/BamHI restriction sites. The PCR product obtained was digested with XhoI/BamHI and cloned into the vector pIRES-EGFP (Invitrogen) to which the FLAG (DYKDDDK) epitope was fused in frame just after the signal peptide cleavage site and the V5 (GKPIPNPLLGLDST) epitope placed at the C terminus of the molecule. All other point mutants of SKI-1 and rSt-1 were generated with the QuikChange II site-directed mutagenesis kit (Stratagene) using the above constructs as template and appropriate pairs of oligonucleotides (Table 1): I985A (S2/AS2), M990A (S3/AS3), M990L (S4/AS4), M990I (S5/AS5), I989L (S6/AS6), Y994A (S7/AS7), N995A (S8/AS8), Y/N994/995A (S9/AS9). All recombinant cDNAs constructs were confirmed by DNA sequencing.


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  		TABLE 1
Oligonucleotides used for site-directed mutagenesis

 
Recombinant Stump-2 (rSt-2) and Cytosolic Tail (rCT)—The smaller C-terminal fragments starting at Met990 (aa 990-1052) and rCT (aa 1023-1052) were PCR-amplified using SKI-1-V5 as template and the oligonucleotide pairs S10/AS10 and S11/AS11, respectively. The PCR products obtained were digested with either XhoI/BamHI or PstI/AgeI and cloned into pIRES-EGFP, and their DNA was sequenced in both directions. The other two constructs, r{Delta}CT and rBTMD, were previously described (15).

Biosynthetic Analysis—HEK293 cells (2-4 x 105) in 60-mm dishes were transiently transfected with 1.2 µg of SKI-1-V5 cDNA using Effectene (Qiagen). Two days post-transfection, the cells were washed and pulse-labeled with 400 µCi/ml [3H]Arg (Amersham Biosciences) for 4 h (19). The cell lysates were immunoprecipitated with mAb/V5 (1:500) in buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 6.8), 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and a mixture of protease inhibitors (Roche Applied Science). The immunoprecipitates were resolved by SDS-PAGE on 8% Tricine gels, dried, and autoradiographed as described (20).

Western Blot Analysis—To detect the presence of St-2, HEK293 cells were transiently transfected with either the wild-type or active site mutant H249A cDNA constructs, as mentioned previously. CHO-K1 and SRD-12B cells were transiently transfected with 4 µg of SKI-1 or SKI-1 mutant cDNAs using Lipofectamine 2000 (Invitrogen). Two days post-transfection, cells were washed twice with phosphate-buffered saline and lysed in SDS buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1% SDS) containing a mixture of protease inhibitors. The samples were incubated on ice for 30 min, and the lysates were run on 8% Tris-Tricine gel. Following SDS-PAGE, proteins were transferred to nitrocellulose membrane (Amersham Biosciences) that was subsequently analyzed by immunoblotting using mAb/V5 (1:5000), and the protein bands were visualized by an ECL chemiluminescence kit (Amersham Biosciences), used according to the manufacturer's instructions.

Effect of Various Classes of Protease Inhibitors on the Generation of rSt-2—4 x 105 CHO-K1 cells were transiently transfected with 4 µg of SKI-1-V5 cDNA and rSt-1. 24 h post-transfection, the cells were washed and incubated for 6 h in medium containing different protease inhibitors (21). Western blot analyses were carried out following 8% SDS-PAGE using mAb/V5 (1:5000). Inhibitors were tested at various concentrations (Table 2). Thus, the inhibitors were directed against (i) metalloproteases and matrix metalloproteases (EDTA (200 µM), Captopril (100 µM) (Sigma), tissue inhibitors of metalloproteinase TIMP1 (150 µM) and TIMP2 (180 µM), phosphoramidon (10 µM), tumor necrosis factor-{alpha} protease inhibitor TAPI (100 µM) (Peptides International Inc.), and GM6001 (25 µM) (Chemicon International Inc.)); (ii) serine/cysteine protease inhibitors (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (300 µM), polyarginine (2 µM), zinc sulfate (ZnSO4) (50 µM) (Sigma), soybean trypsin inhibitor (15 µM), leupeptin (100 µM) (Roche Applied Science), N{alpha}-p-tosyl-L-lysine chloromethyl ketone (100 µM), aprotinin (10 µM), phenylmethanesulfonyl fluoride (10 µM), and chymostatin (100 µM) (Roche Applied Science)); (iii) proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-choloromethylketone (10 µM) (Bachem); (iv) aspartic protease pepstatin (1 µM); (v) signal peptide peptidase inhibitor (Z-LL)2-ketone (100 µM) (Calbiochem); (vi) proteasome inhibitor N-acetyl-leucinal-leucinalnorleucinal (ALLN) (2 µM) (Sigma); (vii) cAMP-dependent protein kinase inhibitor phorbol 12-myristate 13-acetate (10 µM); (viii) {gamma}-secretase inhibitors {gamma}-XVIII (10 µM), {gamma}-IX (10 µM) (Calbiochem), and {gamma}-secretase inhibitor (100 µM) (Enzyme Systems Product DFK-167); and (ix) brefeldin A (BFA) (5 µM) (Calbiochem). Some of the protease inhibitors were analyzed following co-transfection (e.g. in CHO-K1 and Neuro 2A cells) with rSt-1 cDNA and protease nexin-1, Spn4.1A wild type (WT), Spn4.1 RRLL mutant, {alpha}1-antitrypsin, {alpha}1-antichymotrypsin, {alpha}1-PDX cDNAs at a ratio of 1:4 (substrate/inhibitor). 48 h post-transfection, cells were washed and resolved on an 8% Tris-Tricine gel. Detection by Western blotting was done using mAb/V5 (1:5000).


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  		TABLE 2
Protease inhibitors used for inhibition studies (21)

 
N-terminal Microsequencing Analysis of rSt-2—Transiently transfected HEK293 cells were pulse-labeled for 4 h with 250 µCi/ml [35S]Met or 350 µCi/ml [3H]Tyr (Amersham Biosciences). Lysates were immunoprecipitated with mAb/V5 (1:500), and proteins were resolved by SDS-PAGE on 8% Tris-Tricine gel, immobilized to nitrocellulose PSQ membrane (Millipore). The radiolabeled rSt-2 protein was excised and micro-sequenced as previously described on an Applied Biosystems Procise cLC protein sequencer (20).

Mass Spectrometric Analyses—CHO-K1 and HEK293 cells were transfected with either pIRES-V5 vector control, SKI-1-V5, or rSt-1-V5 cDNAs. 48 h post-transfection, samples were immunoprecipitated using mAb/V5 (1:500). All of the bound antibody-antigen complex was eluted from Protein A beads by incubation in 2 x 150 µl of 0.1 M glycine (pH 2.8) for 10 min at room temperature with shaking. Supernatants were collected, combined, and neutralized with 30 µl of 1 M Tris-HCl (pH 9.0). The elutants were concentrated 20x with an Amicon Ultra YM3 Centricon (Millipore Corp., Bedford, MA) and equilibrated in 0.1% trifluoroacetic acid. For time-of-flight mass spectrometric analysis, 10 µl of the sample was applied to an Au Chip (Ciphergen Biosystems Inc., Palo Alto, CA) and allowed to air-dry at room temperature. 1 µl of saturated sinapinic acid in 50% acetonitrile plus 0.5% trifluoroacetic acid was added to each spot. Mass spectrometric analysis was performed by time-of-flight mass spectrometry on a Ciphergen Protein Biology System II (PBS II). Analyses represent an average of 100 shots, and masses were calibrated internally with All-in-1 Protein Standards (Ciphergen Biosystems Inc., Palo Alto, CA).

In Vitro Transcription/Translation—rSt-1 was excised from pIRES-rSt-1-V5 using HindIII/BamHI and subcloned into the pCDNA3 vector. The mutants M990A and I989L were obtained using the Quikclone mutagenesis kit (Stratagene). Recombinant pCDNA3-rSt1-V5 (WT), M990A and I989L cDNAs were transcribed/translated using a TNT coupled reticulocyte system according to the manufacturer's protocol with wheat germ extract (Promega) containing [35S]Met in the presence or absence of canine pancreatic microsomal membranes. The reaction was carried out at 30 °C for 90 min, samples were separated on SDS-PAGE, and Western blots were analyzed using mAb/V5 (1:5000).

Production of Human SKI-CT Antibody—Human SKI-1 polyclonal antibody was generated by immunizing two rabbits with a peptide containing an N-terminal Cys linked to the segment aa 1036-1051 of SKI-1 (C-RPQLMQQVHPPKTPSV), which was conjugated to keyhole limpet hemocyanin using a standard keyhole limpet hemocyanin conjugation kit protocol (Sigma). The two rabbit bleeds were analyzed, and the best one (R2-02) called Ab-CT was used for Western blot analysis (1:1000) and subcellular localization (1:500) studies.

Immunocytochemistry and Confocal Microscopy—To detect endogenous SKI-1, human hepatocyte (HuH7) cells were plated, and 24 h later the cells were fixed with 4% formaldehyde for 30 min at room temperature and washed with phosphate-buffered saline containing 0.1% Triton to permeabilize them. Immunostaining was done as described previously (22). Cells were incubated with primary Ab/CT antibody (1:500) at 4 °C overnight along with markers for different compartments cis/ medial Golgi (Golga1) (1:500), early endosomal marker (EEA1) (1:500), and late endosomal marker (cation-independent mannose 6-phosphate receptor; CI-MPR) (1:500) (Abcam) at 4 °C overnight. The cells were washed and incubated with a mixture of two secondary fluorescently labeled antibodies (i.e. anti-rabbit (Alexa Fluor 555) or anti-mouse (Alexa Fluor 647) (Invitrogen) at room temperature for 1 h). Samples were analyzed on a Zeiss LSM-510 confocal microscope. HuH7 cells (4 x 105 cells) were transiently transfected with SKI-1-V5, rSt-1, rSt-2, {Delta}CT, rBTMD (15), and rCT constructs. 24 h post-transfection, the permeabilized cells were treated and analyzed as above.

Immunocytochemistry of SKI-1 at the Cell Surface—For cell surface labeling, we used nonpermeabilizing conditions (23). HuH7 cells were transiently transfected with 4 µg of SKI-1 cDNA, and 24 h later the cells were labeled with the fluorescent CT-B conjugate as recommended (Vybrant Lipid Raft Labeling Kit; Molecular Probes). The cells were then washed three times with 1x phosphate-buffered saline fixed with 4% paraformaldehyde for 1 h at 4 °C. After fixation, the cells were washed again and incubated overnight with 1:500 Ab/N (15) and then incubated with a secondary fluorescently labeled Alexa Fluor 555-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h at room temperature.

Effects of rSt-1 and rSt-2 on the Processing of ATF6 and Tunicamycin on the Generation of St-2—4 x 103 CHO-K1 cells were co-transfected with 1 µg of rATF6 and empty 3 µg of pIRES2 vector (control) or with 3 µg of cDNAs coding for SKI-1, rSt-1, its mutant I989L, or rSt-2. In another experiment, we used triple transfections of 1 µg each of rATF6 and SKI-1, along with either an empty 2 µg of pIRES2 vector (control) or 2 µg of cDNAs coding for either rSt-1 or rSt-2. 24 h later, cells were treated or not with 2 µg/ml tunicamycin for 12 h. Thereafter, the cells were treated with ALLN (Sigma) at a final concentration of 25 µg/ml for 1 h. The lysates were resolved on 6% SDS-PAGE and analyzed by Western blot using either a 1:5000 dilution of anti-FLAG M2 monoclonal antibody (Stratagene), as reported (6, 24), or mAb/V5 at a 1:5000 dilution.


Figure 1
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  		FIGURE 1.
Schematic representation of human SKI-1/S1P and the generation of St-2. A, schematic representation of human SKI-1 with respective autocatalytic processing sites. The arrows point to the positions of the signal peptide, A, B'/B, and C cleavages, and shedding site. The fragments generated after each event are indicated with numbered amino acids. The recognition sites of N-terminal Ab/N (aa 634-651) and C-terminal Ab/CT (aa 1036-1051), mAb/V5 antibodies used for immunoblotting and immunocytochemistry analysis, are depicted. B, the leftmost panel shows the biosynthetic analysis of HEK293 cells transiently transfected with SKI-1-V5 and labeled with [3H]Arg. Cell lysates were immunoprecipitated and run on 8% Tris-Tricine gels and autoradiographed. The other panels represent Western blot analysis of HEK293, SRD-12B, and CHO-K1 transiently transfected with SKI-1, H249A, and shedding site mutants L952A, K948A/L952A/L953A (KLL/A), and K948A. 48 h post-transfection, cell lysates were resolved on SDS-PAGE, and the immunoreactive SKI-1-containing proteins were revealed using mAb/V5. The arrowheads point to the migration positions of SKI-1, St-1, and St-2. The percentage generation of St-2 is indicated at the bottom of each gel. These values are representative of similar independent experiments repeated more than five times.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of the C-terminal Products of SKI-1—Following transient transfection in three cell lines of C-terminally V5-tagged full-length SKI-1 (SKI-1), we analyzed the processing of the membrane-bound zymogen proSKI-1. HEK293 cells were pulse-labeled with [3H]Arg for 4 h, the cell lysates were immunoprecipitated with mAb/V5, and the products were analyzed by SDS-PAGE in Tris-Tricine. The reported membrane-bound active ~106-kDa SKI-1 (Fig. 1A) autocatalytically sheds itself to produce a soluble secreted ~98-kDa soluble SKI-1 (15), leaving behind a membrane-bound ~13-kDa stump (St-1) (Fig. 1B), as reported (15). However, in this more resolving separation, we also observed a smaller ~8.5-kDa V5-positive product running at the bottom of the gel, estimated at 11-17% of total (St-2; Fig. 1B), suggesting that it represents a shorter C-terminal fragment. To further probe the possible cell type dependence of the generation of St-2, we analyzed the SKI-1 products in two other cell lines, namely SRD-12B (lacking SKI-1 expression) (18) and CHO-K1. These cells were transiently transfected with cDNAs coding for WT SKI-1, its active site mutant (H249A), and its shedding site mutants (L952A, KLL/A, and K948A) (15). Cell lysates were analyzed by Western blotting using a mAb/V5, revealing the presence of St-1 and/or St-2 in all cells (Fig. 1B). In addition, although the autocatalytic generation of St-1 is abrogated in cells expressing the inactive enzyme SKI-1-H249A and in the triple shedding site mutant KLL/A (15), we observed that in all cases, the St-2 fragment was generated at a level of ~15%. Furthermore, unlike the autocatalytic shedding event leading to the production of St-1, the generation of St-2 is an SKI-1 activity-independent process. This is based on the presence of this fragment in both SRD-12B cells and the three cell lines expressing the inactive SKI-1-H249A (Fig. 1B). Based on our calculations, we can now state that in CHO-K1 cells St-2 is ~2-fold better generated from the rSt-1 than from full-length SKI-1 or its H249A mutant (Fig. 2, A and B). The estimated percentage generation of St-2 from FL-SKI-1 and its H249A mutant are comparable in CHO-K1 cells and somewhat lower in HEK293 cells. Since these values are representative of at least five experiments, we trust that this phenomenon is cell type-dependent. We note that the ratio of St-1 to St-2 is much higher in HEK293 cells than in CHO-K1 cells. The reason behind this is not clear. The results also demonstrated that shedding into St-1 is not a prerequisite for the generation of St-2, since its production was observed in all shed site mutants that result in reduced (L952A and K948A) or absent (KLL/A) St-1 as well as in SKI-1-H249A (Fig. 1B).

Inhibition of St-2 Production—In order to identify whether the generation of St-2 is protease-dependent, we overexpressed WT SKI-1 (Fig. 2A) or a secretable form of St-1 containing a signal peptide followed by a FLAG tag at the N terminus and a V5 tag at the C terminus (rSt-1; Figs. 1A and 2B) in CHO-K1 cells. Two days post-transfection, we incubated the cells expressing SKI-1 (Fig. 2A) with either various protease inhibitors targeting different classes of enzymes (Table 2) (21) or the general serine protease inhibitor AEBSF (21) or brefeldin A (BFA), a fungal metabolite that leads to the dissociation and fusion of most of the Golgi apparatus with the ER (25). In addition, we expressed either WT or SKI-1-H249A in M19 cells lacking the expression of site-2 protease S2P (17). We also co-expressed rSt-1 with general protease inhibitors, namely protease nexin-1 (26), Spn4.1A WT and its RRLL derivative Spn4.1A-RRLL (27), {alpha}1-antitrypsin, {alpha}1-antichymotrypsin, and the furin/PC5 inhibitor {alpha}1-PDX (19, 28) in CHO-K1 and Neuro-2a cells (Fig. 2B). Cell lysates were analyzed by Western blot using mAb/V5. Although all inhibitors did not affect the production of St-2, we confirmed the earlier observation that AEBSF inhibits SKI-1 (21) and hence the autocatalytic generation of St-1 (Fig. 2A, cross). In Fig. 2A, we note that ALLN, a proteasome and an inhibitor of Ca2+-dependent lysosomal cysteine proteases (29), increases mostly the level of St-1 but not really St-2 (30% compared with 25% for WT). Since we later show that St-1 and St-2 as well as SKI-1 sort to endosomes/lysosomes, it is possible that St-1 is degraded by lysosomal proteases, including Cys-proteases, and the latter are inhibited by ALLN, which is not as specific a proteasome inhibitor as lactacystine. Similarly, EDTA seems to decrease the production of St-1, possibly by its ability to bind calcium, which would reduce the activity of SKI-1 (4, 15). In view of its ~8.5-kDa size and likely membrane attachment, it was possible that the signal peptidase may be the cognate-converting enzyme. However, incubation with the signal peptidase inhibitor (Z-LL)2-chloromethylketone (30) had no effect on St-2 production (Fig. 2A, right). However, we noticed that only BFA treatment resulted in a major reduction or complete inhibition of the production of St-2 either from its inactive H249A mutant (Fig. 2A, star) or resulting from expression of rSt-1 (Fig. 2B, star). Altogether, the available data suggest that St-2 production can occur in the ER and is abrogated by BFA treatment. Since BFA was never reported to be a protease inhibitor but could inhibit protein synthesis in cultured cells (31), we were faced with the possibility that either BFA may bring in from the Golgi an inhibitor or St-2 may not be generated by proteolysis but rather may be the result of an alternate mechanism that is inhibited by BFA. However, since the only known Golgi-resident protease inhibitor PN1 (26) does not affect St-2 production (Fig. 2B), this suggests that the latter hypothesis is more likely.


Figure 2
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  		FIGURE 2.
Inhibition of the generation of St-2. Different classes of protease inhibitors were tested for their ability to inhibit the production of St-2. A, CHO-K1 cells were transiently transfected with SKI-1 or H249A. 24 h post-transfection, cells were washed and incubated with different protease inhibitors for 6 h as described under "Experimental Procedures." Following treatment, the cell lysates were resolved on 8% Tris-Tricine gels, and the immunoreactive proteins were revealed using mAb/V5. Indicated by a cross is the effect of AEBSF on the production of St-1; the star points to the BFA treatment, which inhibits the production of St-2. B, co-transfection of rSt-1 cDNA (aa 954-1052) along with various known serine protease inhibitors in CHO-K1 and Neuro2A cells at a ratio of 1:4 (substrate/inhibitor). 48 h post-transfection, the cell lysates were run on 8% Tris-Tricine gels and analyzed using mAb/V5. The star indicates the inhibition of St-2 in cells treated with BFA. The percentage generation of St-2 is indicated at the bottom of each gel. These values are representative of similar independent experiments repeated more than five times. PMA, phorbol 12-myristate 13-acetate; SBTI, soybean trypsin inhibitor; TLCK, N{alpha}-p-tosyl-L-lysine chloromethyl ketone; PMSF, phenylmethanesulfonyl fluoride.

 
N-terminal Sequencing and Mass Spectrometric Analysis of St-2—Since the level of St-2 obtained from full-length SKI-1 is rather low (Fig. 1B), and in order to isolate enough protein for microsequencing, HEK293 cells were transiently transfected with the C-terminally V5-tagged rSt-1 (Fig. 1A). Two days post-transfection, the cells were labeled with either [35S]Met or [3H]Tyr for 4 h, the lysates were immunoprecipitated with mAb/V5, and the proteins were separated by SDS-PAGE. Microsequencing data of the generated St-2 revealed the presence of Met at positions 1 and 22 and Tyr at position 5, demonstrating that the St-2 protein starts at Met990 of SKI-1 (see sequence in Fig. 3A). To determine the molecular mass of St-2, we generated enough of this protein from 5 x 107 transiently transfected HEK293 and CHO-K1 cells with cDNAs of SKI-1 or rSt-1 and analyzed by time-of-flight mass spectrometry the molecular masses of immunoprecipitated products (Fig. 3B). The data showed the presence of an ~8.5-kDa St-2 protein obtained from both cell lines with an experimental molecular mass of 8472.7-8473.0 Da. Based on a calculated theoretical mass of 8466.8 Da (lacking the N-terminal Met990), we concluded that most of the St-2 represents the segment (aa 991-1052 + 14-aa V5). However, microsequencing data of radiolabeled St-2 indicated that it starts at Met990, probably reflecting the difference between the de novo synthetic product that still retains some of the N-terminal Met990 and the steady state protein, which lost the Met990, probably by an aminopeptidase, which recognizes to a lesser extent the Met-Pro991 sequence (32, 33). Accordingly, the generation of St-2 could be due to cleavage at DIPGGI989 {downarrow} M990PGRYN (Fig. 3A) or result from alternative translation starting at Met990.


Figure 3
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  		FIGURE 3.
Sequencing and mass spectrometric analysis. A, HEK293 cells were transiently transfected with rSt-1 cDNA and labeled for 4 h with [35S]Met or [3H]Tyr. Cell lysates were immunoprecipitated with mAb/V5 and resolved on an 8% Tris-Tricine gel. The deduced sequence positions of the radiolabeled residues are shown. B, mass spectrometric analysis of SKI-1 cleavage products. Shown are time-of-flight mass spectrometric analyses of the molecular masses of the proteins immunoprecipitated with mAb/V5 from cell lysates of HEK293 and CHO-K1 cells overexpressing either the control empty vector, rSt-1, or SKI-1-V5. The data show a peak at 8473.0 Da following the sequence PGGIM990 {downarrow} (experimental 8472.7-8473.0 Da versus theoretical 8466.8 Da).

 
Identification of Met990 as a Critical Amino Acid by Mutagenesis and in Vitro Transcription/Translation Studies—To define the important amino acids involved in the generation of St-2, different mutant constructs of rSt-1 were obtained using site-directed mutagenesis (Fig. 4A): I985A, I989L, M990L, M990A, M990I, Y994A, N995A, and the double mutant Y994A/N995A (Fig. 4A). These rSt-1 constructs were expressed in CHO-K1 cells, and lysates were analyzed by Western blot using mAb/V5 to detect the generation of St-2 (Fig. 4B). Mutants of Met990 to leucine, alanine, and isoleucine failed to generate St-2 (Fig. 4B, stars), suggesting the critical importance of Met990 for the generation of St-2.

In order to test whether Met990 represents a start codon for an alternatively translated transcript, we mutagenized the nucleotide present at positions -3 from the ATG (ATCATG to CTCATG), which would create a less favorable Kozak sequence (34) and result in an I989L mutation. The data clearly show that the latter mutation resulted in >90% reduction in the generation of St-2 (Fig. 4B, star), whereas all other mutants had no effect (Fig. 4B). An independent confirmation of this observation was sought using the full-length protein. Accordingly, analysis of the WT and mutated M990A SKI-1 revealed that in both HEK293 and CHO-K1 cells, the M990A mutation completely blocked the generation of St-2 from SKI-1 (Fig. 4C). The absolute requirement of methionine for St-2 generation strongly suggested that this product was a result of alternate translation starting at Met990. We therefore generated constructs in the pcDNA3 vector of WT and the M990A and I989L mutants of rSt-1 (Fig. 4A). These constructs were transcribed and translated in vitro using TNT Quick Coupled Transcription/Translation systems using a wheat germ extract in the presence or absence of canine microsomes, as recommended by the manufacturer (Promega). Microsomes were added to enable efficient cleavage of the signal peptide, membrane insertion, and translocation ensuring higher translation efficiency. The reaction products were separated on an 8% Tris-Tricine gel, and the proteins were transferred to a nitrocellulose membrane and immunoblotted using mAb/V5 (Fig. 4D). The data clearly show the presence of St-2 in the WT, which is reduced by >70% in the I989L mutant and completely absent in the M990A mutant (Fig. 4D). Thus, all available data support the conclusion that St-2 is produced by alternate translation. Since it lacks a signal peptide, rSt-2 is expected to have an opposite membrane orientation compared with SKI-1 or rSt-1. It was therefore of interest to define the subcellular localization of rSt-2 compared with either that of endogenous SKI-1 or its recombinant WT SKI-1 and rSt-1.


Figure 4
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  		FIGURE 4.
Mutation analysis identifies the critical amino acid Met990 for the generation of St-2. A, schematic representation of rSt-1 (aa 995-1052) with the signal peptide and N- and C-terminal recognition tags highlighting the different mutants generated. B, Western blot analysis of CHO-K1 cells transiently transfected with the rSt-1 cDNA construct revealed with mAb/V5. Mutation of amino acid Met990 into Leu, Ala, and Ile completely inhibited the generation of St-2, whereas Ile989 inhibited it by almost 90% as indicated by stars. C, CHO-K1 and HEK293 cells transfected with SKI-1 or its M990A mutant. Analysis by Western blotting using mAb/V5 demonstrates the failure to generate St-2 in both cell lines. D, in vitro transcription/translation of WT and its mutants M990A and I989L cloned into pcDNA3 vector. These constructs were transcribed/translated in vitro with wheat germ extract in the presence and absence of canine microsomes. The products were separated on a 8% Tris-Tricine gel, transferred onto a nitrocellulose membrane, and revealed with mAb/V5. The percentage generation of St-2 is indicated at the bottom of each gel. These values are representative of similar independent experiments repeated at least three times.

 
Generation and Analysis of a Specific C-terminal Tail SKI-1 Antibody—To study the subcellular localization of SKI-1-WT, rSt-1, rSt-2, and various recombinant constructs (Fig. 1A), we synthesized the 17-mer peptide C1037RPQLMQQVHPPKTPSV1052, representing the 16 aa of the C terminus of hSKI-1 linked to an N-terminal cysteine residue, which was conjugated to the carrier protein keyhole limpet hemocyanin. This peptide antigen was injected into two rabbits, and the serum from each bleed was collected. The properties of the best resulting polyclonal antibody (Ab/CT, R2-02) were compared with those of mAb/V5 by Western blot and immunocytochemistry. Western blot analysis of transiently transfected human hepatocytes HuH7 and HepG2 cells with the WT cDNA revealed that both antibodies recognize the A, B/B', and C forms of SKI-1 (Fig. 5A). Confocal analysis of the subcellular localization of SKI-1 by immunocytochemistry (22) was performed 24 h after transient transfection of HuH7 cells with WT SKI-1 cDNA and its inactive H249A mutant (Fig. 5B). WT SKI-1 mostly concentrated in paranuclear structures reminiscent of the Golgi (Fig. 5B, arrows), whereas the active site mutant H249A exhibits a diffused perinuclear staining indicating its presence in the ER (Fig. 5B, arrowheads). Thus, Western blot and immunocytochemical analyses give similar results using either mAb/V5 or polyclonal Ab/CT.

Subcellular Localization of Endogenous SKI-1 and Its Recombinant Constructs—Western blot analysis of endogenous SKI-1 in HepG2 and/or HuH7 cells revealed similar A, B/B', and C forms (Fig. 6A). Subcellular localization studies in HuH7 cells (Fig. 6B) revealed that SKI-1 co-localizes with markers of different subcellular compartments (i.e. Golga1 (a cis/medial Golgi marker), EEA1 (an early endosomal marker), and CI-MPR (a late endosomal marker) (22)). Although this agrees with the earlier report on the presence in CHO-K1 cells of endogenous SKI-1/S1P in the cis/medial Golgi (35), our present data further revealed that endogenous SKI-1 can also traffic to endosomes/lysosomes. Upon overexpression of SKI-1 in either HEK293 cells (4) or HuH7 cells (Fig. 6C), we observed similar punctate localizations reminiscent of endosomes/lysosomes (Fig. 6D) (4). However, immunocytochemical analysis of overexpressed SKI-1 using Ab/N recognizing aa 634-651 (15) did not reveal the presence of this enzyme at the cell surface, as compared with the endogenous raft CT-B component cell surface marker (Fig. 6E). These data suggest that the endosomal/lysosomal localization of SKI-1 probably results from a direct transport from the Golgi to endosomes/lysosomes and not via cell surface internalization, as is the case for furin (36).

We next analyzed the localization of overexpressed shorter forms of SKI-1 (Fig. 1A), including rSt-1 (Figs. 6C and 7A) and rSt-2 (Figs. 6C and 7B), r{Delta}CT lacking the C-terminal cytosolic tail but still keeping the transmembrane domain (Fig. 7C) (15), rBTMD, lacking both transmembrane domain and cytosolic tail (Fig. 7D) (15), and the cytosolic tail construct with an added initiator methionine rCT (Fig. 7E). Except for the rBTMD and rCT, localization studies show that all other constructs traffic to the same subcellular compartments as SKI-1 (Figs. 6D and 7). rBTMD is not detectable intracellularly, since it is rapidly secreted (15). Interestingly, rCT that exhibits a putative basic nuclear localization signal (KRRK1032) colocalized with the nuclear marker TOPO3, with a major accumulation within nucleoli (Fig. 7E). The transmembrane domain is common to SKI-1, rSt-1, rSt-2, and r{Delta}CT. It seems to be the dominant domain that determines the localization of these proteins to the Golgi, and subsequent subcellular compartments (i.e. endosomes/lysosomes), since its absence results in a secreted protein that does not accumulate inside the cell.


Figure 5
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  		FIGURE 5.
Comparative analysis of C-terminal polyclonal Ab/CT with mAb/V5. A, schematic representation of human SKI-1 with respective autocatalytic processing sites. The arrows point to the positions of the signal peptide, A, B'/B, and C cleavages. The recognition sequence positions of Ab/N, Ab/CT, and mAb/V5 are shown. B, HuH7 and HepG2 cells were transiently transfected with SKI-1 cDNA. 48 h post-transfection, cells were lysed and run on an 8% glycine gel and revealed using mAb/V5 or Ab/CT. The data show the processing of SKI-1 into A, B/B', and C forms. The pattern of recognition using both antibodies is comparable. C, HuH7 cells transiently transfected with SKI-1 or its active site mutant H249A. The SKI-1 protein was visualized by immunofluorescence by confocal microscopy. Both antibodies reveal that H249A localizes to the ER, whereas SKI-1 localizes to the paranuclear structures reminiscent of the cis/medial Golgi. Bar, 10 µm.

 
Overexpressed rSt-1 or rSt-2 Impede ATF6 Processing by SKI-1—It is well known that treatment of cells with tunicamycin induces ER stress, resulting in increased processing of ATF6 by SKI-1/S1P followed by S2P into a nuclear form (10, 21, 24). In an effort to define a functional role for either rSt-1 or rSt-2, we investigated the effect of either construct on the processing of ATF6 precursor (pATF6) into its nuclear form (nATF6) under stress conditions, such as tunicamycin treatment (10). The data show that co-expression of either rSt-1, its I989L mutant, or rSt-2 with ATF6 reduces its processing into the nuclear form by ~55, 32, and 55%, respectively, whereas SKI-1 increases the level of the latter by ~1.7-fold (Fig. 8, left). St-1-induced reduction of ATF6 processing is ~40% less by the I989L mutant that exhibits a reduced production of St-2. This suggests that St-2 is a better inhibitor of ATF6 processing than St-1. The data also show that co-expression of ATF6 together with SKI-1 and with either rSt-1 or rSt-2, respectively, reduces ~60 and ~72% of the phenotype triggered by SKI-1 alone (Fig. 8, right). In order to test whether stress conditions regulate the alternate translation of SKI-1, resulting in the production of St-2, we analyzed the effect of tunicamycin treatment on the generation of St-2. Accordingly, CHO-K1 cells were transiently transfected with either FL-SKI-1, its H249A mutant (Fig. 9A), rSt-1, rSt-2, and M990A mutant (Fig. 9B) were tested in the absence or presence of tunicamycin. Western blot analysis of the cell lysates revealed that stress induced by tunicamycin does not affect the percentage production of St-2 from either FL-SKI-1, its H249A mutant (Fig. 9A), rSt-1, or rSt-2 (Fig. 9B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Since the discovery of the proprotein convertase SKI-1/S1P (4, 18), it was realized that this convertase can process a number of membrane-bound proteins in the cis/medial Golgi, including transcription factors (2, 3, 11, 24, 37-39) and viral surface glycoproteins (6, 12, 14, 40). Biosynthetic analysis of the zymogen-processing SKI-1 revealed an ordered autocatalytic activation process leading to the formation of intermediate B/B' forms in the ER and then an active C-form in the cis/medial Golgi (4, 15, 41, 42). Indeed, it was soon found that most of the SKI-1 protein localizes in early Golgi compartments in both HEK293 cells (4) and in CHO-K1 cells (35). However, none of these data revealed whether SKI-1 can transiently be localized at other subcellular compartments, which may be implicated in its further processing via autocatalytic shedding into a soluble secreted form (15). It was originally suggested, but not proven, that SKI-1 may be found in punctate structures reminiscent of endosomes/lysosomes and that its concentration could be increased upon neutralizing some lysosomal proteases (4). In this study, we investigated in more detail the trafficking of SKI-1, its autocatalytic shedding into a secreted soluble SKI-1 and a membrane-bound stump St-1, and the generation of a novel membrane-bound form called St-2, independent of SKI-1 activity and/or shedding (Fig. 1).

In agreement with an earlier report (15), we confirmed that SKI-1 activity is needed for its shedding, since both the active site mutant H249A and the SKI-1 inhibitor AEBSF (21, 43) prevented shedding (Figs. 1 and 2). Biosynthetic radiolabeling analysis of either SKI-1 or BTMD-SKI-1-KDEL, which remains in the ER (15), revealed that treatment with brefeldin A or expression of the SKI-1-KDEL construct prevented the production of the St-1 fragment (not shown). This suggests that shedding requires the exit of SKI-1 from the ER and/or acidic conditions found in the cis/medial Golgi. In contrast to the membrane-bound convertases PC7 (44), PC5B (45, 46), and furin (36), which cycle from the cell surface to endosomes, SKI-1 does not seem to localize to the cell surface (Fig. 6E) and hence may directly sort to endosomes/lysosomes (Fig. 6) from the trans-Golgi network. This is in agreement with our observation that shedding is not affected by inhibitors of metalloproteases (Fig. 2 and Table 2), which usually process membrane-bound proteins at the cell surface (16, 47, 48). We deduce that SKI-1 autocatalytic shedding may well occur in the Golgi, where most of the active SKI-1 resides (Fig. 6), and not at the cell surface, where the sheddases are usually localized.


Figure 6
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  		FIGURE 6.
Subcellular localization of endogenous and overexpressed SKI-1. A, to detect endogenous SKI-1, cell lysates from the human cell lines HuH7 and HepG2 were resolved by 6% SDS-PAGE and immunoblotted using Ab/CT. The different forms of endogenous SKI-1, A, B/B', and C forms are indicated. B, immunofluorescence analysis by confocal microscopy of endogenous SKI-1. Merged images of SKI-1 (red labeling), cis/medial Golgi (Golga1), early (EEA1), and late (CI-MPR) endosomal markers (blue labeling) show co-localization of SKI-1 with each of the above markers (pink). C, Western blot using Ab/CT of the lysates of HuH7 cells overexpressing SKI-1, rSt-1, or rSt-2. D, subcellular localization studies using confocal microscopy of overexpressed SKI-1 (Ab/CT; red) and any of the subcellular markers Golga1, EEA1, or CI-MPR (blue). The merged images represent co-localized SKI-1 in the Golgi, early, and late endosomal compartments. Bar, 10 µm. E, cell surface labeling of SKI-1 under nonpermeabilizing conditions. HuH7 cells transiently transfected with SKI-1 were labeled with the red fluorescent CT-B component lipid rafts as cell surface marker and with Ab/N to look for possible SKI-1 localization at the cell surface. Bar, 10 µm.

 
Microsequencing and mass spectrometric analyses demonstrated that St-2 starts at Met990 and consists of the segment comprising amino acids 990-1052 (Fig. 3). Since the production of St-2 is inhibited by BFA treatment (Fig. 2) that can prevent mRNA translation (31) and is abrogated when Met990 is replaced by other amino acids in either the full-length protein or in the rSt-1 construct (Fig. 4), we suspected that St-2 may be produced by an alternate translation of the primary transcript at Met990. In vitro transcription/translation analyses of rSt-1 and its mutants M990A and I989L (Fig. 4D) confirmed that production of St-2 utilizes an internal translation start site at Met990. Although viruses commonly make use of this mechanism to express variant proteins, alternate translation of secretory proteins is rather rare (49). Initiation at Met990 could either be attributed to internal entry of the ribosomal complex, instead of its attachment to the 5' cap, or to leaky ribosomal scanning due to less favorable nucleotide sequence surrounding the initiator methionine. Based on Kozak's rules (34, 50, 51) the consensus sequence around the initiator methionine is GCC GCC (A/G)CC ATG (G/A) (the underlined residues are the most critical). We note that the human SKI-1 nucleotide sequence surrounding Met1 is CTT GTG ACC ATG A, whereas that around Met990 is GGT GGG ATC ATG C. This shows that although both sequences exhibit optimal nucleotides at -3- and -6-positions, the first one has additionally an optimal +4 nucleotide, whereas the second one has a -9 nucleotide. The efficiency of translation is clearly in favor of Met1 (Fig. 1), befitting the predictions of the scanning hypothesis (34, 50, 51). Interestingly, the ATG coding for Met990 is conserved in most vertebrates known to express SKI-1, from Danio rerio to Xenopus tropicalis and Homo sapiens. Since it is found in the last exon XXIV, which is five nucleotides away from the 3' acceptor splice site (52), this may suggest that the C-terminal segment encoded by exon XXIV may have a specific function.

Interestingly, although the membrane orientation of St-2 is expected to be reversed from that of SKI-1 or St-1, they all share the same transmembrane domain, which seems to be critical for their subcellular localization to the Golgi and endosomes/lysosomes (Figs. 6 and 7). A similar critical importance of the transmembrane domain for subcellular localization in the Golgi and possibly endosomes has been reported for the beta-secretases BACE1 (53) and BACE2 (54). Intriguingly, we observed that expression of the highly basic cytosolic tail of SKI-1, lacking acidic residues (4), localizes the polypeptide to nucleoli (Fig. 7E), a subnuclear structure involved in ribosomal RNA gene transcription. Mutation of the stretch KRRK1032 to KAAK1032 or the putative phosphorylation sites S1026A, S1052A, and T12049A did not change such nucleolar localization (not shown). However, the addition of a V5 tag at the C terminus of the cytosolic tail prevented its nuclear localization (not shown), suggesting that the nuclear localization determinants may reside at the C terminus of this sequence.


Figure 7
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  		FIGURE 7.
Immunocytochemical localization of rSt-1 and rSt-2. HuH7 cells were transiently transfected with cDNAs coding for rSt-1 (A), rSt-2 (B), r{Delta}CT (C), BTMD (D), or rCT (E). The cells were fixed, permeabilized, and analyzed for expression of SKI-1 using Ab/CT or Ab/N (red), along with various markers (blue), such as the Golgi marker, Golga1, early endosomal marker, EEA1, late endosomal marker, CI-MPR, or nuclear marker, TOPO3. The presence of pink coloring in merged images demonstrates co-localization of SKI-1 or its fragments with appropriate markers of different compartments. Bar, 10 µm.

 


Figure 8
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  		FIGURE 8.
Effect of rSt-1 and rSt-2 on ATF6 processing. Western blot analysis of ATF6 using anti-FLAG antibody of CHO-K1 cells transiently co-transfected with cDNAs coding for ATF6 and either empty vector (pIRES) or SKI-1, rSt-1, its mutant I989L, or rSt-2 (right) or triply transfected with cDNAs coding for ATF6, SKI-1, and either control pIRES, rSt-1, or rSt-2. 24 h later, cells were treated with 2 µg/ml tunicamycin for 12 h. Thereafter, the cells were treated with 25 µg/ml ALLN for 1 h. The lysates were resolved on 6% SDS-PAGE. The migration positions of the ATF6 precursor (pATF6, ~90 kDa) and its nuclear form (nATF6, ~50 kDa) as well as the percentage cleavages of pATF6 into nATF6 are indicated. These values are representative of similar independent experiments repeated three times.

 


Figure 9
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  		FIGURE 9.
Effect of tunicamycin-induced stress on the generation of St-2. Shown is Western blot analysis using mAb/V5 of CHO-K1 cells incubated in the presence or absence of 2 µg/ml tunicamycin (Tm) and transiently transfected with cDNAs coding for FL-SKI-1 or its active site mutant H249A (A) and rSt-2, rSt-1, or its mutant M990A (B). The samples were analyzed as in Fig. 2. The percentage generation of St-2 is indicated at the bottom of each gel. These values are representative of similar independent experiments repeated three times.

 
Finally, in an effort to define a possible functional role for the observed stumps St-1 and St-2, we checked whether the SKI-1 generated processing of the ER stress response factor ATF6 was affected by the overexpression of either constructs. A representative figure of our three independent experiments shows that both rSt-1 and rSt-2 diminish the processing of pATF6 into nATF6 by an as yet undefined mechanism (Fig. 8, left). Furthermore, we also show that the co-expression of SKI-1 with either rSt-1 or rSt-2 reduces the phenotype triggered by SKI-1 alone (Fig. 8, right). Finally, tunicamycin-induced stress does not seem to increase the level of production of St-1 (Fig. 9), suggesting that the observed phenomenon of alternate translation is not regulated by cellular stress. It is a matter of speculation whether the membrane-bound stumps St-1 and St-2 may interfere with the ER-stress machinery either in the ER or cis/medial Golgi. Alternatively, the rSt-1 and rSt-2 may act as dominant negatives of SKI-1.

In conclusion, the major SKI-1 activity is expected to be mostly in the cis/medial Golgi and may also be found in endosomes. However, although many candidate membrane-bound transcription factor substrates of SKI-1 are known (2, 3, 11, 24, 37-39), the putative endosomal substrates are yet to be identified. Alternatively, the presence of SKI-1 in endosomes/lysosomes may reflect its normal metabolic fate, as was reported for BACE1 (20). It will be interesting to define the determinants of the transmembrane domain that are critical for the subcellular localization of SKI-1 and to possibly separate those required for Golgi localization from the ones needed for endosomal trafficking. Finally, the detailed mechanism behind the possible function of the St-1 and St-2 proteins on ATF6 processing is yet to be unraveled.


   FOOTNOTES
 
* This work was supported by Canadian Institutes of Health Research Grant MOP-36496, by a Canada Chair, and by a private donation from the Strauss Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Ave. W., Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail: seidahn{at}ircm.qc.ca.

2 The abbreviations used are: PC, proprotein convertase; SKI-1, subtilisin kexin isozyme-1; S1P, site-1-protease; ER, endoplasmic reticulum; St-1, stump-1; St-2, stump-2; rSt-1 and rSt-2, recombinant St-1 and -2, respectively; BTMD, before transmembrane domain; rBTMD, recombinant BTMD; CT, cytosolic tail; rCT, recombinant CT; WT, wild type; {alpha}1-PDX, {alpha}1-antitrypsin Portland; CHO, Chinese hamster ovary; Ab, antibody; mAb, monoclonal antibody; Ab/N, anti-SKI-1 N terminus antibody; Ab/CT, anti-SKI-1 C-terminal antibody; BFA, brefeldin A; CI-MPR, cation-independent mannose 6-phosphate receptor; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; ALLN, N-acetyl-leucinal-leucinal-norleucinal. Back


   ACKNOWLEDGMENTS
 
We are indebted to Josée Hamelin, Marie-Claude Asselin, Jadwiga Marcinkiewicz, Ann Chamberland, and Andrew Chen for technical help; Antonella Pasquato and Monica Dettin for peptide synthesis and conjugation; and Nasha Nassoury, Gaetan Mayer, Eric Bergeron, and Annik Prat for constant and valuable advice and help. We gratefully acknowledge the valuable gifts of inhibitors PN-1 (Y. Peng Loh), Spn4.1 and its RRLL mutant (F. Jean), and {alpha}1-PDX (G. Thomas). The secretarial assistance of Brigitte Mary is greatly appreciated.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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