(Received for publication, September 29, 1995)
From the
We have recently described a new serine proteinase inhibitor,
proteinase inhibitor 6 (PI-6). This serpin has features that suggest it
may function intracellularly, but its close resemblance to ovalbumin
serpins like plasminogen activator inhibitor 2 (PAI-2) raises the
possibility that it is secreted to regulate an extracellular
proteinase. To determine whether PI-6 is secreted, we have examined its
cellular distribution by immunohistochemistry and have attempted to
induce its release from platelets and from cultured cells. We find that
PI-6 is present in endothelial and epithelial cells, but it is
apparently cytoplasmic and it is not released from cells in response to
phorbol ester, dibutyryl cAMP or tumor necrosis factor treatment.
It is also not released from activated platelets. The addition of a
conventional signal peptide to the amino terminus of PI-6 directed its
translocation into the endoplasmic reticulum (ER), resulting in
glycosylation but not secretion of the molecule. By contrast, the
addition of the same signal peptide to PAI-2 markedly enhanced its
translocation and secretion. Glycosylated PI-6 was sequestered in the
ER and was incapable of interacting with thrombin. The failure of PI-6
to move along the secretory pathway, and the loss of inhibitory
function of ER-localized PI-6, demonstrates that unlike PAI-2, PI-6 is
not naturally secreted. Taken together, these results suggest that PI-6
has evolved to fulfil an intracellular role and that it represents a
new type of cellular serpin.
Serine proteinase inhibitors (serpins) are a family of structurally related proteins that regulate the activity of serine proteinases involved in extracellular processes such as coagulation, fibrinolysis, complement fixation, and embryo implantation. Several members of the family have lost proteinase inhibitory function and have evolved extracellular functions such as serving as lipophilic molecule transporters and peptide hormone precursors(1) .
Recently,
we and others have identified a new Arg-serpin known as proteinase
inhibitor 6 (PI-6), ()the placental thrombin inhibitor, or
the cytoplasmic antiproteinase(2, 3) . Although PI-6
efficiently inhibits the extracellular proteinases plasmin, trypsin,
thrombin, and urokinase in vitro(4) , it is unusual
because it is present in cytosolic extracts, it is not found in the
medium of cultured cells, it lacks a conventional signal sequence, and
it is sensitive to oxidation(2, 5) . These properties
suggest that PI-6 may have an intracellular function. At present, the
only serpin with a clearly defined intracellular role is the viral
protein crmA, which is an inhibitor of granzyme B and the
interleukin-1
-converting enzyme(6, 7) .
PI-6
closely resembles the ovalbumin serpins. This group of proteins
includes ovalbumin, plasminogen activator inhibitor 2 (PAI-2), the
squamous cell carcinoma antigens (SCCA-1 and SCCA-2), maspin, and the
monocyte neutrophil elastase inhibitor(8, 9) . All of
the ovalbumin serpins lack conventional signal sequences, yet they are
found as extracellular glycoproteins. At least two of these ovalbumin
serpins, PAI-2 and SCCA, appear to exist mainly as cytosolic proteins
but are efficiently secreted and glycosylated in response to specific
stimuli. For example, glycosylated PAI-2 is released from monocytes in
response to tumor necrosis factor and phorbol ester
treatment(10) , and SCCA is released from transformed squamous
epithelial cells(11) . Thus it cannot be inferred from the lack
of a conventional signal sequence and an apparent cytosolic location
that PI-6 is confined intracellularly or that it has an intracellular
function.
To determine if PI-6 is released to function in the
extracellular milieu, we have examined its cellular distribution using
immunohistochemistry and have attempted to induce its secretion from
cultured cells and platelets. Furthermore, we have provided it with a
conventional signal sequence to assess whether it can be efficiently
glycosylated and released if directed into the secretory pathway. We
find that PI-6 is located in endothelial cells, in platelets, and in a
subset of epithelial cells but that it is not released from activated
platelets nor from cultured cells in response to tumor necrosis factor
, phorbol ester, or cAMP analogues. PI-6 directed into the
secretory pathway is glycosylated but loses inhibitory activity and is
retained in the endoplasmic reticulum. On the basis of these studies,
we conclude that PI-6 is not naturally secreted and that it is a true
intracellular serpin.
The PAI-2 expression plasmid pEUKPAI-2 (a gift of Dr. R.
Medcalf) consists of the human PAI-2 cDNA cloned into pEUK-C1
(Clontech). A PAI-2 derivative containing the HA signal sequence was
constructed in a similar manner to pSVHA/PI-6. PCR primers
5`-ATGGAGGATCCTTGTGTG-3` (sense) and 5`-GGACTAGTTAGGGTGAGCAAAATCT-3`
(antisense) were designed to amplify the coding sequences of PAI-2. The
sense primer inserts a BamHI site near the initiation codon
and substitutes Leu with Pro. The antisense primer inserts
an SpeI site just after the termination codon. Following
amplification with Vent polymerase, the fragment was cloned into
pCR
II for verification, released by BamHI-SpeI digestion and ligated to pSHT cleaved with BamHI and SpeI.
Tunicamycin (10 µg/ml, Boehringer Mannheim) was added to the medium of transfected COS cells 18 h before labeling commenced and was included throughout the labeling procedure.
Figure 1:
Immunohistochemical staining of PI-6
in human dermis. Sections of normal human dermis were stained with
affinity-purified anti-PI-6 polyclonal antibodies. Adjacent sections
incubated with nonimmune serum showed no specific staining (not shown). A, a section of sweat gland in which cells of the coiled
excretory duct, but not those of the secretory portion, were stained
positively for PI-6 (magnification 200). Staining of the
capillary endothelium was also observed (arrow). B,
higher magnification view (400
) showing the intense staining of
the inner layer of cuboidal duct epithelial cells and weaker staining
of the outer layer.
The demonstration of PI-6 staining in endothelial and epithelial cells accounts for the wide distribution of PI-6 previously observed by RNA analysis. The presence of PI-6 in these cells is also consistent with a model for PI-6 function in which it is released by epithelial or endothelial cells to participate in the regulation of extracellular proteinases. In this respect, it might resemble the closely related serpin, PAI-2, which is released to regulate urokinase(10) . To test if PI-6 is normally released following synthesis or on stimulation of particular cells, we examined its production in a number of systems in which regulated or constitutive release might occur.
Figure 2: PI-6 expression in platelets. Platelets collected from human blood were lysed, activated, and fractionated as described under ``Experimental Procedures.'' The presence of PI-6 in platelet fractions or releasate was demonstrated by immunoprecipitable complex formation with thrombin. Aliquots were incubated with iodinated thrombin followed by reduction, 10% SDS-PAGE, and autoradiography. Releasate from activated platelets generated a complex (lane 1) that was not immunoprecipitable with PI-6 antibodies (lane 4). Cytosol from activated platelets generated a smaller complex (lane 3) that was immunoprecipitable with PI-6 antibodies (lane 6). Total lysate from unstimulated platelets generated both complexes (lane 2), but only the smaller species was immunoprecipitable (lane 5).
To test whether PI-6 is released on platelet activation, we stimulated platelets with iodinated thrombin to cause release of the granule contents and then separated the platelets from the releasate. The activated platelets were then subjected to lysis by freeze-thawing to prepare cytosolic extracts, which were incubated with a fresh aliquot of iodinated thrombin. All of the samples were then reduced and analyzed by SDS-PAGE and autoradiography. As shown in Fig. 2(lane 1), the releasate from activated platelets contained the larger complex, which was not immunoprecipitable using anti-PI-6 antibodies (lane 4). By contrast, the cytosol of the activated platelets contained the smaller complex (lane 3), which was immunoprecipitable (lane 6). These results demonstrate that PI-6 is present in platelet cytosol but that its secretion is not induced on platelet activation.
PI-6 Is Not Released by Resting or Stimulated Cultured Cells-We have examined a number of cultured cell lines by RNA analysis, indirect immunofluorescence, or thrombin-complexing assays for the presence of PI-6. These include primary human umbilical vein endothelial cells; the human lines HeLa, HepG2, HT1080, K562, U937, and THP1; the simian line, COS-7; and the murine lines SP2, Balb/c 3T3, F9, E14, and STO. With the exception of THP1, all of these cells produce PI-6 (data not shown). We have tested conditioned media from most of these cells for PI-6 activity by the thrombin complexing assay or for PI-6 antigen by immunoblotting and have not detected any evidence for PI-6 release into the medium.
By analogy to the situation for PAI-2 or SCCA, we
considered the possibility that PI-6 is only released into the medium
in response to a specific signal. To test this, we treated K562 cells,
U937 cells, or COS-7 cells with inducers of the protein kinase C signal
transduction pathway (phorbol 12-myristate 13-acetate) and the protein
kinase A pathway (dibutyryl cAMP). We also treated the cells with tumor
necrosis factor , which is a potent inducer of PAI-2(24) .
Following the treatments, the medium was removed and cytosolic extracts
were prepared. Iodinated thrombin was added to both media and extracts,
and PI-6 antiserum was used to immunoprecipitate any complexes formed.
As shown in Fig. 3, thrombin
PI-6 complexes were detected
in cytosolic extracts of both untreated and treated cells, but not in
any of the media samples. There was no evidence of any increase in the
amount of intracellular complexing forming activity in response to any
of the treatments, suggesting that PI-6 biosynthesis is not stimulated
by these agents. This was supported by RNA analysis, which showed that
PI-6 mRNA levels did not increase in the treated cells (data not
shown).
Figure 3:
PI-6 activity (thrombin complexing
ability) is not detected in media conditioned by agonist-treated cell
lines. Cells were untreated (-ve) or treated for 24 h
with 25 ng/ml phorbol 12-myristate 13-acetate (PMA), 50 ng/ml
tumor necrosis factor (TNF), or 1 mM dibutyryl cAMP. Iodinated thrombin was added to the cell lysates (C) and to the culture supernatant (M) prior to
immunoprecipitation with anti-PI-6 antibodies. Immune complexes were
collected and analyzed by 10% SDS-PAGE and
autoradiography.
Given that PI-6 is inactivated in oxidizing
conditions(5) , it is possible that PI-6 is released from
cultured cells but that functional assays fail to detect it because it
is rapidly inactivated. To test this possibility, we analyzed selected
media samples for PI-6 antigen by immunoblotting, but we were unable to
detect PI-6 protein (data not shown). In addition, we carried out
pulse-chase experiments in COS cells transfected with a PI-6 expression
vector. PI-6-producing cells were starved in media lacking methionine
and then labeled for 30 min with [S]methionine.
After the labeling (pulse) period, complete media were added and the
cells were incubated for specified times (chase). At each time point,
the medium was collected and the cells were lysed. Both media and
lysates were immunoprecipitated with PI-6 antiserum and the immune
complexes analyzed by SDS-PAGE. Lactate dehydrogenase assays carried
out on the samples showed that a negligible degree of nonspecific cell
lysis occurred during the experiment.
As shown in Fig. 4, the predominant protein immunoprecipitated from the cell lysates immediately after the labeling period was a 42-kDa species, as expected for PI-6(5) . Preimmune serum did not recognize this protein (data not shown). The amount of PI-6 present in the cell extracts did not decrease markedly over 10 h, and no release into the medium was detected, suggesting that PI-6 is reasonably stable in the cytosol and that it is not secreted under these conditions. Taken with the experiments on release of PI-6 activity from cultured cells, these results suggest that PI-6 is not normally secreted and may have evolved to function intracellularly.
Figure 4:
Biosynthesis of human PI-6 in transfected
COS cells. COS cells were transfected with pSVTfPTI/P DNA. 48 h
posttransfection, cells were starved for 30 min in media lacking
methionine, labeled for 1 h in media containing 100 µCi of
[S]methionine and then incubated in complete
media for the indicated times. Cell extracts and media samples were
prepared at each time point and immunoprecipitated with PI-6
antibodies. Immune complexes were collected, reduced, and analyzed by
10% SDS-PAGE and fluorography.
Addition of a Signal Peptide to PI-6-Although a simple interpretation of our results is that PI-6 is not a secreted protein, its similarity to PAI-2 and SCCA leaves open the possibility that it is secreted under certain (perhaps rare) circumstances. If this is true, a simple prediction can be made that if directed to the endoplasmic reticulum (ER), PI-6 should be able to travel through the secretory pathway, and that glycosylated PI-6 should retain proteinase inhibitory function. To test this, we decided to efficiently direct PI-6 into the secretory pathway by providing it with a conventional signal sequence.
As shown in Fig. 5, a derivative of PI-6 (HA/PI-6) containing the influenza virus HA signal sequence fused to the amino terminus of PI-6 was constructed by PCR-mediated mutagenesis of PI-6 and in-frame cloning into the expression vector, pSHT(18) . This vector provides the SV40 early promoter followed by the HA signal sequence, cloning sites, and termination codons. A similar derivative of PAI-2 (HA/PAI-2) was constructed as a control (Fig. 5). (Although it is predominantly cytosolic, PAI-2 is known to be capable of travelling through the conventional secretory pathway(25, 26) , and the efficiency with which it enters the ER can be enhanced by attaching a heterologous signal sequence(27) .)
Figure 5: Addition of the HA signal peptide directs PI-6 and PAI-2 into the secretory pathway. Upper panel, diagram shows the amino-terminal sequence of HA/PI-6 and HA/PAI-2 compared to PI-6 and PAI-2. Boxed residues comprise the HA signal peptide. The signal peptidase recognition site for this peptide lies between the glycine and aspartate residues at the end of the boxed region(18) . Lower panel, HA/PI-6, PAI-2, and HA/PAI-2 expression in COS cells. See Fig. 4for experimental details. HA/PAI-2 was immunoprecipitated using a monoclonal PAI-2 antibody. Arrows indicate position of glycosylated PAI-2.
The HA/PI-6, PAI-2, and HA/PAI-2 expression plasmids were transfected into COS cells and subjected to pulse-chase analysis as described above. Entry into the ER and travel through the secretory pathway was predicted to result in an apparent increase in the size of both proteins and release into the medium. Since PI-6 (42 kDa) and PAI-2 (47 kDa) each have three potential N-linked glycosylation sites, increases in size of at least 10-12 kDa were expected for both molecules. As shown in Fig. 5A, proteins approximately 42, 45, 47, and 50 kDa in size were immunoprecipitated from extracts of COS cells expressing HA/PI-6. These probably represent HA/PI-6 glycosylated at 0, 1, 2, or 3 sites, respectively. The number and sizes of these proteins did not alter during a 3-h chase period, and none were detected in the media, suggesting that HA/PI-6 cannot exit the secretory pathway.
By contrast, three forms of PAI-2 were detected in extracts of COS cells immediately after labeling (Fig. 5B). The smallest, most abundant form represents cytosolic, unglycosylated PAI-2 (47 kDa), which is not released into the medium. Three larger forms were present in extracts in much lower amounts and represent glycosylated PAI-2 (50-55 kDa). Slight but increasing amounts of these larger forms were detected in media samples during the chase period (Fig. 5B). This pattern of expression is consistent with the inefficient secretion of PAI-2 that has been described previously(26) . Addition of the HA signal sequence to PAI-2 significantly altered the pattern of expression (Fig. 5C). In this case, far less 47-kDa PAI-2 was observed, and significant quantities of the larger forms were present in the cell extracts and were secreted into the medium. This confirmed that the HA signal can markedly increase the efficiency of PAI-2 entry into the ER, leading to a substantial increase in the amount of PAI-2 that exits the secretory pathway.
Figure 6:
Effect of tunicamycin on HA/PI-6
biosynthesis. COS cells were transfected with pSVTf (Vector),
pSVTfPTI/P (PI-6) or pSVtfHA/PI-6 (HA/PI-6) DNA and
analyzed without(-) or with (+) the addition of tunicamycin
(10 µg/ml). At 48 h posttransfection, cells were starved for 30 min
in media lacking methionine and labeled for 4 h in media containing 100
µCi of [S]methionine. Cell extracts were
prepared and immunoprecipitated with PI-6 antibodies. Immune complexes
were collected, reduced, and analyzed by 10% SDS-PAGE and fluorography. Closed arrow indicates position of native PI-6 or
unglycosylated HA/PI-6. Open arrows indicate different
glycoforms of HA/PI-6.
The failure to detect secretion of the HA/PI-6 glycoforms (Fig. 6) suggested that they are trapped somewhere along the secretory pathway. To assess where this block occurs, indirect immunofluorescence experiments were carried out. COS cells producing either PI-6, HA/PI-6, PAI-2, or HA/PAI-2 were fixed, permeabilized, and probed with either PI-6 or PAI-2 antibodies. After detection with FITC-conjugated secondary antibodies, cells were examined by fluorescence microscopy (Fig. 7). Cells producing PI-6 and PAI-2 showed the diffuse, intracellular pattern of staining expected for cytosolic proteins, whereas cells producing HA/PAI-2 showed the characteristic Golgi staining observed for secreted glycoproteins. By contrast, cells containing HA/PI-6 showed a reticular pattern of staining usually associated with proteins located in the ER.
Figure 7: Intracellular localization of HA/PI-6 by indirect immunofluorescence. COS cells were transfected with pSVTfPTI/P (A), pSVTFHA/PI-6 (B), pEUKPAI-2 (C), pSVTfHA/PAI-2 (D), pSVmHA/NEO (E), or pSVHA/NEO (F). 48 h posttransfection, cells were fixed, permeabilized, and probed with rabbit PI-6 antiserum diluted 1:200 (upper panels), mouse PAI-2 monoclonal antibody diluted 1:50 (middle panels), or rabbit NEO antiserum diluted 1:200 (lower panels). The primary antibodies were detected by the appropriate sheep fluorescein isothiocyanate-conjugated secondary antibodies. Cells were examined by fluorescence microscopy.
To confirm its apparent ER localization, the pattern of HA/PI-6 staining was compared with that seen in COS cells producing HA/NEO, which is a chimeric protein consisting of the HA signal fused to the bacterial enzyme neomycin 3`-phosphotransferase (HA/NEO). It has previously been shown that the HA signal can direct the NEO polypeptide into the ER where it is trapped, whereas mutation of the HA signal sequence results in a protein (mHA/NEO) that is cytosolic(17) . The expression patterns in cells producing HA/NEO and mHA/NEO resembled those of HA/PI-6 and PI-6, respectively (Fig. 7), supporting the proposition that HA/PI-6 is sequestered in the ER.
Glycosylation of nascent proteins is an ordered process that commences in the ER and continues in the Golgi apparatus. Proteins remaining in the ER normally have different oligosaccharide structures compared with those that have travelled to the Golgi and can be distinguished by the effect of endoglycosidase H (endo H). Resident ER proteins or nascent secretory proteins that have not left the ER contain ``high mannose'' oligosaccharides that can be removed by endo H. Proteins that have entered the Golgi apparatus have their N-linked carbohydrates modified and are resistant to endo H. On this basis, it was predicted that HA/PI-6 proteins trapped in the ER would be sensitive to endo H. COS cells producing HA/PI-6 or HA/NEO were metabolically labeled as described above, chased for 0 or 2 h, lysed, and immunoprecipitated using the appropriate antibodies. Immune complexes were split and treated or not treated with endo H prior to SDS-PAGE analysis.
As shown in Fig. 8, endo H treatment of HA/PI-6 immunoprecipitates completely removed the HA/PI-6 glycoforms, and no endo H-resistant proteins were observed 2 h after the labeling was terminated. Similar results were obtained with immunoprecipitates from cells containing the ER-resident HA/NEO protein. These results support the notion that HA/PI-6 is sequestered in the ER, and suggest that little movement of HA/PI-6 from ER to Golgi occurs.
Figure 8:
Effect of endoglycosidase H treatment on
HA/PI-6. COS cells were transfected with pSVTfHA/PI-6 (HA/PI-6) or pSVHA/NEO (HA/NEO) DNA. 48 h
posttransfection, cells were starved for 30 min in media lacking
methionine, labeled for 30 min in media containing 100 µCi
[S]methionine, and then incubated in complete
media for the indicated times. Cell extracts and media samples were
prepared at each time point and immunoprecipitated with the appropriate
antibodies. Immune complexes were collected and treated (+) or not
treated(-) with endo H. Samples were then reduced, and analyzed
by 10% SDS-PAGE and fluorography.
Figure 9:
Assessment of the thrombin complexing
ability of HA/PI-6. COS cells were transfected with pSVTf (Vector), pSVTfPTI/P (PI-6), or pSVtfHA/PI-6 (HA/PI-6) DNA. At 48 h posttransfection, cells were starved
for 30 min in media lacking methionine and labeled for 4 h in media
containing 100 µCi of [S]methionine. Cell
extracts were prepared and analyzed without(-) or with (+)
the addition of thrombin prior to immunoprecipitation with PI-6
antibodies. Immune complexes were collected, reduced, and analyzed by
10% SDS-PAGE and fluorography. Arrow indicates the
thrombin
PI-6 complex.
Loss of complex forming ability could be due to
steric hindrance mediated by the carbohydrate side chains on HA/PI-6 or
to malfolding of the molecule in the ER. Treatment of transfected COS
cells with tunicamycin did not result in the formation of
thrombinHA/PI-6 complexes (data not shown), suggesting that the
loss of inhibitory function is not caused by glycosylation of HA/PI-6.
Our previous studies have shown that PI-6 is an Arg-serpin that is produced in many tissues and most closely resembles a group of proteins collectively known as the ovalbumin serpins(2, 4, 30) . Two of these ovalbumin serpins, PAI-2 and SCCA, are predominantly cytosolic but can be secreted under certain circumstances(8) . This, coupled with the fact that PI-6 efficiently inhibits extracellular proteinases such as plasmin, thrombin, and urokinase, suggested that PI-6 might function outside the cell. Our observation that PI-6 is synthesized by endothelial and epithelial cells is consistent with this idea. However, as discussed below, our failure to detect release of PI-6 under a number of conditions and our demonstration that PI-6 directed into the ER is nonfunctional and not secreted, strongly suggests that PI-6 has an intracellular role.
Most serpins that function extracellularly possess amino-terminal signal peptides that serve to direct entry of the nascent protein into the ER. The ovalbumin serpins are unusual in that secretion of these molecules occurs in the absence of conventional signal sequences. The nature of the signal(s) that direct ovalbumin serpin secretion is poorly understood, but it is thought to comprise sequences in the first and second helices (near the amino terminus)(10) . Although PI-6 resembles the ovalbumin serpins in this region, it is not possible to predict from sequence information alone whether PI-6 is secreted.
The efficiency of these unconventional signals varies markedly, ranging from the ovalbumin signal that directs complete secretion of the molecule, to the one on PAI-2 that does not appear to function until stimulation of PAI-2 biosynthesis greatly increases its intracellular concentration. This variation in efficiency can be explained by Rapoport's model for the interaction of a signal sequence with the signal recognition particle (SRP)(31) , in which this interaction is postulated as an equilibrium between unbound SRP on one hand and the SRP-signal complex on the other. Thus SRP can have different binding affinities for different signals, and in the case of a poor signal, binding to the SRP might not occur until a significant increase in the signal concentration kinetically favors the formation of the SRP-signal complex. Consequently, if PI-6 possesses a weak signal sequence, it can be predicted that increased PI-6 transcription and the biosynthesis of large quantities of PI-6 might be accompanied by constitutive secretion of the molecule. This is certainly the case for PAI-2 produced in phorbol ester-treated U937 cells; PAI-2 transcription increases markedly and is paralleled by secretion of up to 70% of nascent PAI-2 (25) . In this study, we were unable to identify a treatment that increases expression of endogenous PI-6 mRNA or that leads to the release of PI-6 protein. Furthermore, overexpression of human PI-6 in COS cells did not lead to secretion.
An alternative pathway for PI-6 release might be through regulated secretion, in which the molecule is stored in an intracellular compartment and released in response to a specific signal. Although our histological and immunofluorescence experiments provide no evidence for such a compartment, we used platelets to model this situation because they contain PI-6, the regulated release of platelet contents is well-characterized, and they are known to release protease nexin 1 (another serpin) on activation. In addition, we treated several PI-6-producing cell lines with agents designed to activate intracellular signaling pathways likely to trigger regulated secretion. PI-6 was not released from activated platelets, nor was it released from stimulated cell lines, suggesting that regulated secretion of PI-6 does not occur. Another argument against intracellular storage and regulated secretion of PI-6 is that entry of proteins into storage compartments usually occurs via the secretory pathway after movement through the Golgi. Since PI-6 cannot move past the ER, it is unlikely to be stored in a conventional secretory granule.
A number of studies have been performed in which normally cytosolic or nuclear proteins have been introduced into the ER by attaching a heterologous conventional signal sequence(17, 32, 33) . In all cases, the proteins successfully entered the ER and were glycosylated but did not move along the secretory pathway. The reason for this is thought to be a failure to fold correctly due to oxidation and formation of inappropriate disulfide bonds. Malfolded proteins in the ER are retained and degraded by a mechanism that remains obscure(34) . By contrast, heterologous signal sequences added to normally secreted proteins do not impair processing and secretion(17, 27) . On the basis of such studies, we predicted that if PI-6 is a intrinsic cytosolic protein, attachment of the HA signal would result in incorrect folding and failure to exit the ER. On the other hand, if PI-6 can be glycosylated and secreted under certain circumstances, attachment of the HA signal should simply enhance the amount appearing in the medium. Our studies clearly support the first prediction and argue strongly that PI-6 is a cytosolic serpin that has evolved to meet an intracellular function. Given that PI-6 is an inhibitory serpin, it is likely that this involves the regulation of an intracellular proteinase.
Taken with our previous work demonstrating differences between PI-6 and the ovalbumin serpins in gene localization and structure(20, 35) , the results of this study show that PI-6 can now be distinguished from the ovalbumin serpins by three criteria: gene structure, gene localization, and the failure to exit the secretory pathway. The recent finding that the MNEI gene co-localizes with PI-6 on human chromosome 6p25(36, 37) indicates that MNEI may not belong to the ovalbumin serpins as suggested previously(8) . If this is the case, it is conceivable that MNEI will have a gene structure similar to PI-6, and will prove to be nonsecreted. Thus PI-6 may be the prototype of a new class of intracellular serpins.