©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Propeptide Is Nonessential for the Expression of Human Cathepsin D (*)

Suzanne C. Fortenberry , John M. Chirgwin (§)

From the (1) Research Service, Audie L. Murphy Veterans Administration Medical Center and the Departments of Medicine and Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

When the 44-amino acid propeptide of human procathepsin D was deleted by mutagenesis in vitro, the mature protein was stably expressed and secreted from transfected mammalian cells. The secreted protein was correctly folded as judged by its binding to pepstatinyl-agarose. We were unable to detect lysosomal targeting of the propeptide-deleted protein, and targeting was not restored by the substitution of the propeptides from pepsin or renin. We conclude that its propeptide is not essential for the folding of nascent cathepsin D. Efficient lysosomal targeting in mammalian cells appears to require the precursor form of the molecule.


INTRODUCTION

Cathepsin D, an abundant, soluble hydrolase, is a member of the aspartic proteinase gene family, which includes the secretory proteins pepsin, chymosin, and renin (Tang and Wong, 1987), all of which share substantial amino acid sequence identity. The mature enzymes show a bilobed structure flanking a large active site cleft (Davies, 1990) which, in the precursor form, is occluded by an approximately 45-amino acid propeptide. Although not as highly conserved as the mature sequences of the enzymes, the propeptides also show structural similarities and several invariant residues (Foltmann, 1988; Koelsch et al., 1994).

Unlike the other members of the aspartic proteinase family, which are mostly secretory proteins, procathepsin D is sorted to the lysosome. Before reaching the Golgi apparatus, procathepsin D is modified so that the oligosaccharides linked to asparagines 70 and 199 bear mannose 6-phosphates. Upon exiting the Golgi, procathepsin D is sorted to a prelysosomal/late endosomal compartment by binding to mannose 6-phosphate receptors. As the pH in this compartment drops, the receptors dissociate and recycle, whereas the ligand is delivered to the primary lysosome (Kornfeld, 1990; Erickson, 1989). Procathepsin D can also follow a mannose 6-phosphate-independent pathway to the lysosome in some cell types (Glickman and Kornfeld, 1993).

Once committed to the lysosome, human procathepsin D undergoes three proteolytic maturation steps: cleavage of its 44-amino acid propeptide, removal of a COOH-terminal dipeptide, and digestion of a six-amino acid loop around residue 100 (Erickson, 1989; Hasilik, 1992; Delbrück et al., 1994). A 46-kDa form, lacking the propeptide, occurs in both late endosomes and the primary lysosome as a biosynthetic intermediate. When procathepsin D is overexpressed, some of the 52-kDa form of the protein exits the cell via the constitutive secretory pathway (Hasilik, 1992).

High resolution structures are known for many mature aspartic proteinases (Davies, 1990), including cathepsin D (Metcalf and Fusek, 1993; Baldwin et al., 1993), but pepsinogen is the only gene family member for which a precursor structure has been published (James and Sielecki, 1986; Hartsuck et al., 1992). In pepsinogen, relative to pepsin, two differences are apparent: 1) the active site cleft is occupied by the propeptide, and 2) the amino terminus moves about 40 Å from its position in the precursor form, upon activation, replacing one of the strands in the connecting -sheet, which was previously contributed by the NHterminus of the propeptide. The major folded domains and the active site cleft itself show no gross conformational changes. Crystallographic data are unavailable for the precursor forms of renin or cathepsin D, but a striking feature of all the aspartic proteinases is their high degree of tertiary structural similarity (Davies, 1990). The conservation of the protein folding pattern in the gene family has made it practical to create useful computer graphics models of aspartyl proteases for which sequence but not crystallographic data are available (Hutchins and Greer, 1991). Thus Hsueh and Baxter (1991) have suggested for prorenin that the precursor folds into a three-dimensional structure very similar to that of pepsinogen. Procathepsin D also probably shares substantial structural similarity to pepsinogen.()

All of the characterized aspartic proteinases are synthesized via the endoplasmic reticulum/Golgi pathway and are exposed to similar folding conditions and chaperones (Nilsson and Anderson, 1991). It is thus likely that all members of the family follow a similar pathway of protein folding in vivo. Active renin has been successfully expressed from transfected mammalian cells, after deletion of its propeptide sequence by mutagenesis in vitro, by a number of groups (Chidgey and Harrison, 1990; Chu et al., 1990; Norman et al., 1992; Rothwell et al., 1993). Pepsinogen can be reversibly refolded following denaturation, whereas conditions for successful folding of mature pepsin activity in the absence of its propeptide have not been found, despite extensive efforts (Ahmad and McPhie, 1978; Pain et al., 1985; Lin et al., 1993). These results suggest that some, but probably not all, aspartic proteinases can fold efficiently in the absence of their propeptides. Thus renin is unlike bacterial -lytic protease, whose large propeptide sequence functions as a specific template for the productive folding of the mature polypeptide chain (Baker et al., 1992). A number of other secreted subtilisin-related proteases also require their propeptides to fold correctly (Shinde and Inouye, 1994).

Conner (1992) reported that an imperfect deletion of the propeptide sequence of human procathepsin D abrogated stable expression of the transfected protein in mammalian cells. He also demonstrated that attachment of the propeptide to the NHterminus of -lactalbumin did not direct the fusion protein to lysosomes. We wondered if an exact deletion of its propeptide would permit mature cathepsin D to fold directly, when expressed in the endoplasmic reticulum of mammalian cells, and if this deletion would impair targeting to the lysosome, since Baranski et al. (1992) have shown that the propeptide sequence may contribute determinants for mannose 6-phosphate modification in Xenopus oocytes.

We therefore constructed and expressed in rodent and human cell lines a mutant of human procathepsin D with a precise deletion of the propeptide. Overexpression resulted in secretion of a correctly folded protein capable of binding to the active site reagent pepstatin. We were unable to detect delivery to the lysosome of the propeptide-deleted cathepsin D. Targeting was not restored by substitution of the propeptide from human pepsin or renin.


MATERIALS AND METHODS

Enzymes for molecular biology were from New England Biolabs (Beverly, MA) and used according to the manufacturer's instructions. Radiochemicals were from DuPont NEN, tissue culture reagents from Life Technologies, Inc., and biochemicals from Sigma. Oligodeoxynucleotides were synthesized by the Biopolymer Sequencing and Synthesis Facility, Department of Biochemistry and used without further purification, following deblocking and lyophilization.

Expressed aspartic proteinase polypeptides were immunoprecipitated by standard procedures (Harlow and Lane, 1988; Faust et al., 1987), followed by electrophoresis on denaturing, reducing 12.5% polyacrylamide gels (Laemmli, 1970), and fluorography with ENHANCE (DuPont NEN) according to the manufacturer's instructions. Autoradiographic data were quantified by digitized densitometric analysis using a Macintosh 840AV computer with video camera and National Institutes of Health Image 1.55 software (Wayne Rasband, National Institutes of Health, Bethesda, MD).

Cathepsin D was immunoprecipitated with a rabbit polyclonal antibody prepared against the mature human enzyme purified from placentas.() Rabbit polyclonal antibodies against human pepsinogen A and recombinant human prorenin were generously supplied by Dr. I. M. Samloff (Sepulveda Veterans Administration Hospital, Los Angeles, CA) and Dr. P. M. Hobart (Pfizer Central Research, Groton, CT). Protein molecular weight markers for gel electrophoresis were labeled with [C]formaldehyde (ICN, Irvine, CA) by reductive methylation (Jentoft and Dearborn, 1979).

All of the protein constructs tested here carry a widely used 13 amino acid epitopic extension (Kolodziej and Young, 1991) derived from the human c- myc gene and recognized by an available monoclonal antibody (Evan et al., 1985), which permits affinity purification of labeled mutant protein from transfected cells (Sachdev et al., 1991). It can also be used to monitor delivery of cathepsin D to the lysosome, since the myc epitope is proteolytically removed there (Pelham, 1988). We have shown (Sachdev et al., 1991)that both human (293) and rodent (CHO)() cells efficiently express myc-extended procathepsin D (CDM) and prorenin in transient transfection assays. Expressed proteins were stable for 24 h, as shown by pulse-chase experiments.Fra et al. (1993) have demonstrated that COOH-terminally extended versions of CDM can be retained in the ER lumen. The COOH-terminal epitopic extension offers a probe of the proteolytic environment to which nascent aspartic proteinase precursors are exposed, without altering intracellular sorting. Horst et al. (1993) fused lysozyme to the COOH terminus of procathepsin D to monitor delivery to the lysosomes of CHO cells.

Mutagenesis in vitro followed the procedure of Kunkel et al. (1987). Single-stranded template DNAs were purified from phagemids grown in Escherichia coli CJ236 supplemented with uridine. Phagemids were precipitated twice with polyethylene glycol before deproteinization. Mutagenic oligonucleotides consisted of complementary anchors flanking the mutated region (Sambrook et al., 1989). The double-stranded DNA products were transformed into E. coli strain XL1-B, and the resultant colonies screened by filter hybridization with P-end-labeled mutagenic oligonucleotides. Filters were repetitively washed at increasing temperatures and autoradiographed to identify clones with no mismatches to the oligonucleotides (Sambrook et al., 1989). Mutants were verified by restriction mapping and partial DNA sequencing (Trevino et al., 1993). DNAs for transfection into mammalian cells were purified by the polyethylene glycol precipitation protocol described in Sambrook et al. (1989) and used without CsCl banding.

The control version of procathepsin D, whose expression is driven by a cytomegalovirus promoter, is referred to as procathepsin D-myc (CDM), a 52-kDa bis- N-glycosylated, full-length procathepsin D, with its carboxyl terminus extended by a 13-amino acid myc epitope (Sachdev et al., 1991). The expressed fusion protein was originally constructed by Pelham (1988). A parallel version of human prorenin with the myc extension has been described elsewhere.The 44-amino acid propeptide sequence (residues 44 through 1) of human procathepsin D was deleted from CDM by mutagenesis in vitro (Kunkel et al., 1987) with a 30-mer oligonucleotide complementary to the last 5 codons of the prepeptide and the first 5 codons of the mature sequence (Faust et al., 1985). Cathepsin D mutants lacking the glycosylation site at Asnhave been described.() These were combined with the 44/1 mutation by ligating fragments of the single mutants via a unique FspI restriction site at base pair 775 of the cDNA. A series of six mutants with the propeptides interchanged with the bodies of the mature proteins of CDM, pepsinogen, and RNM were constructed with 30-mer oligonucleotides and an overlap extension PCR mutagenesis procedure (Ho et al., 1989). We use the cathepsin D numbering convention of Faust et al. (1985), in which the propeptide is numbered from 44 to 1.

Tissue culture of human 293 and Chinese hamster CHO-L76 cells, transfection with DNAs, labeling with [S]methionine, and immunoprecipitations have been described.The 293 (human embryonal kidney) cell line was obtained from the ATCC. CHO-L76 cells (Cockett et al., 1991) were from Celltech Ltd. (Berkshire, United Kingdom). Both 293 and CHO-L76 cells express an adenoviral E1a gene product to enhance transcription from the cytomegalovirus promoter (Gorman et al., 1990). Cells were transiently transfected by CaPOprecipitation (Gorman et al., 1990). Transfection efficiencies were reproducibly greater than 50% as monitored by the inclusion of 10% pSV2--gal DNA and assaying aliquots of cell lysates for -galactosidase activity (Gorman, 1985). Two days after transfection, the cells were changed to Dulbecco's modified Eagle's medium minus methionine for 2 h, then supplemented with 100 µCi/ml [S]methionine overnight, or as indicated in the figure legends. Medium was removed and cell lysates prepared by three freeze-thaw cycles in the presence of 0.2% Triton X-100. All samples were treated with a protease inhibitor mix (Faust et al., 1987) and immunoprecipitated.

Binding to pepstatinyl-agarose was carried out by standard means (Conner, 1989).


RESULTS

When cathepsin D with its 44-amino acid propeptide deleted (CDM44/1) was expressed in human 293 cells, it was abundantly secreted, at a level about one-third that of the undeleted protein, CDM (Fig. 1). This same moderate reduction of expression is seen when human preprorenin cDNA is transfected in mammalian cells with its propeptide deleted (Chidgey and Harrison, 1989; Chu et al., 1991; Norman et al., 1992; Rothwell et al., 1993).


Figure 1: Cathepsin D with its propeptide deleted is secreted from mammalian cells. Human kidney 293 cells transfected with DNAs expressing procathepsin-myc, CDM ( lanes 1) or CDM44/1 ( lanes 2) were labeled overnight and immunoprecipitated proteins from cell lysates and conditioned media analyzed.



Both secreted intact procathepsin D-myc and propeptide-deleted proteins bound efficiently to pepstatinyl-agarose (Fig. 2), indicating correct folding of the active site. Immunoprecipitation of the fractions unbound to pepstatinyl agarose showed that the majority of secreted cathepsin D polypeptides, with or without the propeptide attached, had bound to the column (data not shown).


Figure 2: Secreted CDM and 44/1 both bind efficiently to pepstatinyl agarose. Conditioned media containing CDM ( lanes 1 and 2) or CDM44/1 (lanes 3-5) were prepared as in Fig. 1. Lanes 1 and 3 show total labeled proteins in the media. Lanes 2 and 4 show material specifically bound to pepstatinyl agarose at pH 3.6 and eluted at pH 8.5. Lane 5 shows the pepstatinyl agarose unbound material. Samples were not immunoprecipitated.



Human cells cannot readily be used to assay lysosomal targeting of human procathepsin D, because of the inability to distinguish between endogenous and transfected proteins. However, rodent cells express a single chain (46 kDa) enzyme, whereas lysosomally processing the transfected human protein to a two-chain (31 + 14 kDa) form (Conner et al., 1989; Horst and Hasilik, 1991). Xenopus oocytes do not process human procathepsin D to this two-chain form (Faust et al., 1987). When we transfected CDM44/1 into rodent CHO-L76 cells, we were unable to detect any lysosomal delivery of the human protein in multiple experiments ( Fig. 3and data not shown). Isidoro et al. (1991) have shown that transfected human procathepsin D does not compete efficiently for targeting to the lysosome with the endogenous precursor in hamster BHK cells. Therefore we considered it important also to test the lysosomal targeting of human propeptide-deleted cathepsin D in human cells.


Figure 3: Propeptide-deleted cathepsin D does not target to the lysosome. The experiment was the same as in Fig. 1, except CHO-L76 cells were used.



We combined the 44/1 mutation with ones at the second N-linked glycosylation site Asn. These latter mutants are not blocked in lysosomal targeting (since the oligosaccharide at Asnstill supports the mannose 6-phosphate-mediated pathway) but can be distinguished from the endogenous human protein.Loss of glycosylation lowers the apparent Mof the human heavy chain from about 31,000 to 29,000 (Horst and Hasilik, 1991). When the mutants N199A, N199D, and N199S were combined with 44/1, no targeting to the lysosome was detectable in transfected human 293 cells (Fig. 4). The data in Fig. 4and in similar experiments (not shown) were analyzed densitometrically. In those lanes, such as lanes 4 and 5, where the mutant did not target to the lysosome, the background in the region where the targeted band should have been seen was always less than 5% of the density seen for a successfully targeted mutant. Thus, in these experiments targeting was reduced at least 20-fold, which was the limit of detection in the experiment with the highest background. The mutant N199T also gave the same result (not shown). The secreted, nontargeting mutants appear to be of somewhat higher Mthan expected. We are presently testing whether this is the result of modification of the oligosaccharide to the complex type in the Golgi apparatus.


Figure 4: Lysosomal targeting of monoglycosylated forms of procathepsin D is abrogated by deletion of the propeptide. Transfected 293 cells were analyzed as in Fig. 1. A shows cell lysates and B the corresponding conditioned media. Lanes 1 and 2 are mock-transfected and CDM positive controls. Lane 3 is CDM N199D, which removed the second glycosylation site. Lanes 4-6 combine the 44/1 propeptide deletion with mutations at Asn-199; lane 4 is N199S, lane 5 is N199A, and lane 6 is N199D. Successful delivery to the lysosome of an N199X mutant protein results in the appearance of a 29-kDa cathepsin D heavy chain band.



To ascertain if the cathepsin D propeptide carried lysosomal targeting determinants, cathepsin D, with or without an N199T mutation, was expressed with its propeptide sequence replaced by that from human pepsin or human renin. No lysosomal targeting in the absence of the cathepsin D propeptide sequence was detected (Fig. 5 A). The mutant proteins were expressed productively as judged by the stable secretion of precursors into the conditioned media (Fig. 5 B). The decreased mobility of the secreted protein in lane 8 (cathepsin D with the propeptide of renin) may be the consequence of altered oligosaccharide processing at Asn.


Figure 5: Cathepsin D targeting to the lysosome requires its own propeptide sequence. Transfected 293 cells were analyzed as in Fig. 1. A shows cell lysates, and B shows the corresponding conditioned media. Lane 1 is CDM positive control. Lanes 2 and 3 are N199D and N199T mutants, which target to the lysosome. Lanes 4 and 5 are CDM44/1 and CDM44/1,N199A, which do not target to the lysosome. Lanes 6 and 7 are CDM N199T with the cathepsin D propeptide replaced by the propeptide of human renin or human pepsinogen, respectively. (The sample for lysate ( lane 6) was lost during handling.) Lanes 8 and 9 correspond to lanes 6 and 7, but without the mutation at Asn-199. Lane 10 is the mock-transfected negative control.




DISCUSSION

When human preprorenin cDNA is transfected in mammalian cells with its propeptide sequence deleted, mature active protein is expressed at levels only moderately below those of the wild-type proenzyme (Chidgey and Harrison, 1989; Chu et al., 1991; Norman et al., 1992; Rothwell et al., 1993). We thus anticipated that similarly deleted procathepsin D could be expressed successfully in mammalian cells. This prediction was confirmed by the results shown in Fig. 1. The reduction in the level of expression of procathepsin D relative to cathepsin D was similar to that seen in the prorenin:renin experiments. It is possible that this reduction reflects a decrease in the efficiency of folding of nascent polypeptides in the endoplasmic reticulum in the absence of their propeptides.

The propeptide-deleted (44/1) protein bound efficiently to pepstatinyl agarose (Fig. 2). Such binding has been used as a criterion of correct folding for the expressed protein, both precursor and mature forms (Conner and Udey, 1990), as well as for chimeras between procathepsin D and pepsinogen (Glickman and Kornfeld, 1993). Our results (not shown) indicated that the majority of the secreted, propeptide-deleted cathepsin D was productively folded. The data in Figs. 1 and 2 suggest that the propeptide of cathepsin D facilitates protein folding in vivo but is not essential for the process.

We were initially concerned that the propeptide-deleted cathepsin D might display some proteolytic activity during its sorting within acidic intracellular compartments. Such activity could be deleterious for the expressing cells. When we combined the 44/1 deletion with the conversion of the essential active-site Asp-33 to Ser, there was no change in the expression pattern seen in Fig. 1(data not shown). A parallel active site mutation in pepsinogen abolished catalytic activity, without perturbing folding of the active site cleft, as judged by successful binding of the mutant pepsinogen to pepstatinyl agarose (Lin et al., 1989).

Conner (1992) expressed a mutant of procathepsin D with most of the propeptide sequence removed. The encoded protein retains its 20-amino acid signal peptide, followed by 8 amino acids which replace the first 2 residues of the mature protein. This 8-amino acid replacement consists of LVRI (the first 4 residues of the propeptide) plus RNSG. The construct which we have reported here fuses the signal peptide directly to residue +1 of the mature protein. Conner's mutant protein was unstable when expressed in mammalian cells. In pepsinogen the first 6 residues of the propeptide sequence form the first strand of a six-stranded antiparallel -sheet connecting the two major lobes. Upon activation to pepsin, this strand is replaced by the mature amino terminus (James and Sielecki, 1986; Hartsuck et al., 1992). The positions of the first 12 residues of the mature sequence differ greatly in the crystal structures of pepsinogen and pepsin. The first 10 or so residues of both pro- and mature forms of other aspartic proteinases are probably also constrained by the requirement to fold into the six-stranded -sheet. We think it likely that Conner's mutant, whose sequence was dictated by available restriction enzyme sites in the DNA sequence, violated the constraints on this region of the cathepsin D protein sequence. Richo and Conner (1994) have recently shown that mutation of residues Leu, Ile, or Valin the human cathepsin D propeptide does not block expression or targeting to the lysosome in mouse Ltkcells.

Sagherian et al. (1994) and Tao et al. (1994) have expressed propeptide deletions of the lysosomal enzymes -hexosaminidase B and cathepsin L, respectively, in mammalian cells. In neither case was the deleted protein able to complete folding or exit the endoplasmic reticulum. However, both mutants were constructed using restriction sites in the cDNA sequences, so that the proteins retained some residues from their precursor amino termini.

The observation by van den Hazel et al. (1993) that formation of active proteinase A in yeast requires co-expression of the propeptide in cis or in trans may reflect requirements for this sequence in vacuolar targeting (Klionsky et al., 1988) and not directly in protein folding. It is also possible that the propeptide region of the endogenous cathepsin D in our mammalian cells transfected with the CDM44/1 vector is similarly able to catalyze folding in trans. This, however, leaves unexplained the abrogation of lysosomal targeting by deletion of the propeptide.

Baranski et al. (1990, 1992) have concluded that the major lysosomal targeting determinant (as assayed in Xenopus oocytes) of cathepsin D lies in the carboxyl-terminal lobe, in particular Lys-203 and residues 265-292, although lesser contributions are made by the amino-terminal portion, including the propeptide, as well (Cantor et al., 1992). Glickman and Kornfeld (1993) tested many of the mutants used by Baranski et al. (1990, 1992) and Cantor et al. (1992) in Epstein-Barr virus-transformed B lymphocytes from a patient with I-cell disease (phosphotransferase deficiency). They concluded that the propeptide sequence did not contribute to lysosomal targeting in this system. From these results propeptide-deleted cathepsin D would be expected to target to the lysosome.

We were thus surprised by the results in Fig. 3 that showed deletion of the propeptide blocked targeting of the protein to the lysosome in transfected Chinese hamster cells. To confirm this observation, we combined the 44/1 deletion with a series of mutationswhich eliminate heavy chain glycosylation at Asn-199. Deletion of the propeptide from the monoglycosylated mutants confirmed that the propeptide was necessary for efficient targeting to the lysosome. Failure to reach the lysosome was not the result of protein instability, since these double mutants expressed well and were secreted into the medium of the transfected cells. Lysosomal targeting was depressed to the limit of detection, at least 20-fold, by the deletion of the propeptide. A series of lysine to glutamic acid mutations in the carboxyl-terminal lobe of procathepsin D was without effect on lysosomal targeting in transfected fibroblasts,suggesting that the failure of the propeptide deletion to target in not an artifact of the mammalian cell system.

Conner (1992) demonstrated that attachment of the cathepsin D propeptide to secretory -lactalbumin did not convert the fusion to a lysosomal protein, although the folding of the propeptide in this construct is unknown. Horst et al. (1993) fused lysozyme to the carboxyl terminus of wild-type human procathepsin D. When expressed in CHO cells, the fusion protein was transported to the lysosome and cleaved. The lysozyme partner had acquired mannose 6-phosphate modification on an introduced N-linked glycosylation site. The results of Conner (1992) and Horst et al. (1993) are compatible with a model in which the major determinants of lysosomal targeting are carried on the surface of mature cathepsin D rather than primarily on the propeptide. We were therefore puzzled why the presence of the propeptide was necessary for lysosomal targeting in the experiments presented here. In view of the conformational differences between the precursor and mature forms of aspartic proteinases, discussed above, it is possible that the propeptide does not itself carry significant targeting information, but rather the targeting information resides primarily in the mature protein sequence but is only correctly configured when the protein is in the proenzyme conformation.

In an attempt to restore the zymogen conformation to cathepsin D deleted of its propeptide, we replaced the cathepsin D propeptide by the similar propeptides from human pepsinogen A and human prorenin. These peptides show less sequence identity than the mature proteins, although they conserve a number of critical residues and the capability to form several -helical segments (Foltmann, 1988; Koelsch et al., 1994). Fusek et al. (1991) have shown that the human cathepsin D propeptide is a strong inhibitor of bovine and chicken pepsins, whereas the chicken pepsin propeptide showed no binding to bovine cathepsin D. These data suggested that interchanging the propeptides of aspartic proteinases by mutagenesis in vitro should be practical for some combinations. When we carried out this experiment (Fig. 5), we observed that the renin propeptide permitted folding and secretion of the mutant protein but lysosomal delivery was not restored. The propeptide from pepsinogen was less effective in promoting expression of the fusion protein, consistent with the results of Fusek et al. (1991).

Our results suggest that the targeting to the lysosome of human procathepsin D in mammalian fibroblasts requires determinants in the propeptide sequence. Such targeting determination occurs in the routing of proteinase precursors to the vacuole in yeast (Klionsky et al., 1988; Valls et al., 1990). The modification of certain blood clotting factors depends on propeptide sequence determinants which signal -carboxylation in a pre-Golgi compartment (Furie and Furie, 1991). Mannose 6-phosphate addition could proceed by a similar mechanism in mammalian fibroblasts, whereas Xenopus oocytes (Baranski et al., 1990, 1992) may recognize different features of human procathepsin D than those which are important in the homologous targeting assay we have used here.


FOOTNOTES

*
This work was supported by Merit and Associate Career Research Scientist awards (to J. M. C.) from the Veterans Administration. 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: Division of Endocrinology and Metabolism, Dept. of Medicine, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7877. Tel.: 210-567-4900; Fax: 210-567-6693.

J. Schorey, H. Lueke, F. A. Quiocho, and J. M. Chirgwin, unpublished data.

Schorey, J., Fortenberry, S. C., and Chirgwin, J. M. (1995) J. Cell Sci. 108, in press.

The abbreviation used is: CHO, Chinese hamster ovary.

Fortenberry, S. C., Schorey, J., and Chirgwin, J. M. (1995) J. Cell Sci. 108, in press.


ACKNOWLEDGEMENTS

We thank Drs. P. M. Hobart, R. T. Taggart, and I. M. Samloff for generously supplying antibodies and cDNA clones, Deepali Sachdev for helpful discussions, and Richard Sunvison for assistance with the pepstatinyl agarose binding experiments.


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