* Diabetes Research Center and Division of Endocrinology, Albert Einstein College of Medicine, Bronx, New York 10461; Research Institute Neurosciences, Vrije Universiteit Faculty of Biology, Amsterdam, The Netherlands; § Department of
Genetics and Development, Columbia University, New York
In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated
secretory pathway. Using mouse pancreatic islets and
the INS-1 -cell line as models, we have compared the
itineraries of procathepsins L and B, two closely related
members of the papain superfamily known to exhibit
low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after
pulse labeling INS cells, a substantial fraction of both
proenzymes exhibit regulated exocytosis. After several
hours, much procathepsin L remains as precursor in a
compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form
and can no longer be secreted. However, in islets from
transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining
within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are
present in immature
-granules, while immunolabeling for cathepsin L, but not B, persists in mature
-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to
occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the
context of different systems, MPR-mediated sorting of
lysosomal proenzymes occurs to a variable extent
within the trans-Golgi network and is continued, as
needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.
Lysosomes are digestive organelles found in all eukaryotic cells (Kornfeld and Mellman, 1989 To date, the trafficking of lysosomal prohydrolases has
been well studied only in cells that produce few or no
secretory granules. From these studies, the basic schema
of posttranslational carbohydrate modification and recognition by mannose-6-phosphate receptors (MPRs)1 is now
generally accepted (von Figura and Hasilik, 1986; von Figura, 1991). By contrast, two substantially different kinds of
hypotheses, which are not mutually exclusive, have prevailed concerning the sorting of regulated secretory proteins. The "sorting for entry" theories include the possibility of one or more conserved receptors whose primary
function would be to bind lumenal content proteins in the
TGN and "usher" them (Kelly, 1987 In the The presence of clathrin coats on IGs implicates the
presence of transmembrane receptors that cluster and bud
from this compartment, during which C-peptide may be
conveyed nonspecifically as a fluid-phase marker. Interestingly, recent work has indicated that the AP-1 clathrinassociated protein complex interacts with the cytosolic tails of MPRs (Glickman et al., 1989 These considerations have raised several important
questions. First, because tunicamycin affects all newly synthesized glycoproteins, including putative sorting receptors, what is the ability to interpret the fate of procathepsin
L after generalized inhibition of N-linked glycosylation?
Second, knowing that there are several cell types in pancreatic islets, can Antibodies and Other Materials
Immunoprecipitating antisera against rat ProB (Rowan et al., 1992 Preparation and Labeling of Cell Lines and Islets
The INS-1 cell line (generously provided Dr. P. Halban, Institut Jeantet,
University of Geneva) was cultured as described (Neerman-Arbez and
Halban, 1993 Islets or cells were lysed in boiling 1% SDS. Samples were then diluted
1:10 in immunoprecipitation buffer yielding final concentrations of: 100 mM NaCl, 1% Triton X-100, 0.2% Na deoxycholate, 0.1% SDS, 10 mM
EDTA, 0.7 mM DTT, 10 mM iodoacetamide, and 25 mM Tris, pH 7.4. An
antiprotease cocktail of aprotinin (1 mU/ml), leupeptin (0.1 mM), pepstatin (10 mM), EDTA (5 mM), and diisopropylfluorophosphate (1 mM) was
added to the islet lysates and collected media. Both cells and media were
precleared by a brief incubation with Zysorbin followed by spinning in a
microfuge before further analysis.
SDS-PAGE, Fluorography, and Phosphorimaging
Immunoprecipitated lysosomal enzymes were resolved by SDS 12%PAGE for cathepsins B and L, while immunoprecipitated insulin was analyzed by SDS 15%-PAGE plus urea using a Tricine buffer system (Schagger and vonJagow, 1987 Ultrathin Cryosectioning of Mouse Pancreatic Islets
Mouse islets prepared as above were fixed for 2 h at room temperature in
a mixture of 0.5% glutaraldehyde and 2% formaldehyde, freshly prepared
from paraformaldehyde, in 100 mM Na-phosphate buffer, pH 7.4. The
fixed islets were embedded in 10% gelatin in PBS and cut into 1-mm3
blocks. These blocks were impregnated with 2.3 M sucrose and further
prepared for cryosectioning as described (Slot et al., 1988 A Significant Fraction of ProB Enters and Then Exits
the Regulated Secretory Pathway of INS Cells
The trafficking of ProB, a high-affinity ligand for MPRs,
was examined in the well-differentiated
Normal Entry but Deficient Exit of ProL in the
Regulated Secretory Pathway of INS Cells
We next used INS cells to examine the trafficking of ProL,
a low-affinity ligand for MPRs (Dong and Sahagian, 1990
To characterize the ultimate fate of intracellular cathepsin L, we compared early chase times to those prolonged
up to 1 d to allow maximum opportunity for newly synthesized ProL molecules to reach their proper final targets.
Unstimulated and stimulated secretions from INS cells
were collected from either 0-4 h of chase or 20-24 h of
chase. A short exposure of the media (Fig. 4, top) demonstrated a fivefold stimulated release of ProL during the
early chase period (Fig. 4, first and third lanes). In the cell
lysates, the arrival of labeled cathepsin L in lysosomes was
modest, based on the appearance of the processed mature
forms (Fig. 4, bottom, first and third lanes). Remarkably,
by 24 h of chase, the newly synthesized mature lysosomal
cathepsin L had almost entirely turned over, while intracellular ProL was still detected in a pool that appeared
longer lived (Fig. 4, bottom, second and fourth lanes). When
the INS cells were exposed to secretagogue during the last
4 h of the experiment, the signal was diminished in proportion to the amount of labeled ProL remaining, but an approximately fivefold stimulated release of ProL was still
obtained (Fig. 4, middle), and the fraction of the remaining
ProL that exhibited stimulus-dependent exocytosis was
not diminished from that seen at the earlier chase time.
Moreover, the disappearance of mature L forms, with the
sustained presence of ProL, suggested that ProL was contained in a compartment from which delivery to lysosomes
was not actively ongoing. Taken together, these findings indicated that a considerable fraction of ProL persisted in
secretory granules.
Is the Correctly Delivered Fraction of ProL to
Lysosomes Conveyed by a Non-MPR Mechanism?
Although only a minority of ProL ever arrives in lysosomes in INS cells, we sought to determine whether this
modest fraction, some of which apparently traverses the
IG compartment, uses MPRs to escape from the regulated
secretory pathway. This issue seemed especially important
since there is evidence that the propeptide of ProL can interact with a putative receptor in intracellular membranes
that can assist in sorting ProL from prelysosomes to lysosomes (McIntyre and Erickson, 1991 In the experiment shown in Fig. 5, INS cells were pulse
labeled and chased for 3.5 h before a 1-h period of stimulus-dependent secretion was analyzed. The left panels
show the results from control cells that are similar to Fig.
3, insofar as partial delivery to lysosomes was detected.
The right panels were performed in parallel from INS cells
treated with tunicamycin. In this case, samples were intentionally double loaded to enhance the sensitivity of detection of mature cathepsin L forms. Nevertheless, it was clear that the arrival of unglycosylated L in lysosomes was
profoundly inhibited, while there was a commensurate increase in the amount of intracellular ProL. Evidently, in
these cells, despite the low affinity of ProL for MPRs, that
portion of ProL which is successfully targeted to lysosomes still uses a carbohydrate-dependent sorting mechanism. Moreover, the increase in missorted ProL after tunicamycin treatment resulted in a proportionate increase in
the pool of unglycosylated precursor in the regulated secretory pathway, as demonstrated by markedly increased stimulus-dependent secretion (Fig. 5, right).
Immunoelectron Microscopy of ProB and ProL in
Mouse Islet Our data obtained in INS cells as well as mouse pancreatic
islets (Kuliawat and Arvan, 1994 In studying the distribution of immunoreactive cathepsins in pancreatic
Similar double labelings confirmed the presence of cathepsin L immunoreactivity in IGs, while substantial persistence of this labeling was observed in many granules that
no longer contained proinsulin immunoreactivity (Fig. 10).
Indeed, mature
Stimulated Secretion of ProB from the
Islets of Transgenic Mice Which Lack the
Cation-dependent MPR
Most lysosomal proenzymes use MPRs to traffic from the
biosynthetic pathway to endosomes, for ultimate delivery
to lysosomes (Kornfeld and Mellman, 1989 Because CI-MPRs can compensate to a large degree for
the absence of CD-MPRs (Ludwig et al., 1994 Therefore, islets from CD-MPR-deficient or control
mice were pulse labeled and chased in the absence of
secretagogues for a sufficient period to allow the labeled
proinsulin and ProB to chase into mature granules and lysosomes, respectively. Upon subsequent analysis of stimulus-dependent secretion, MPR-deficient mice exhibited a
clear increase in stimulated discharge of ProB along with
insulin from mature
Newly Synthesized Prohydrolases Can Exit the
Biosynthetic Pathway at Either the TGN or IGs in
Pancreatic Regulated secretory tissues and cell lines do have a bona
fide TGN compartment (Tooze and Huttner, 1990
In this report, we have tried to examine the dynamic nature of lysosomal proenzyme sorting in pancreatic Despite differences in MPR affinity, a significant fraction of both prohydrolases initially gain access to the regulated secretory pathway in INS cells (Figs. 1-3). However,
with further time, the regulated secretion of ProL persists
in parallel with insulin (Fig. 4), while ProB is selectively
chased out of compartments exhibiting stimulus-dependent
exocytosis (Fig. 1), just as has been observed in normal
mouse pancreatic islets (Kuliawat and Arvan, 1994 From a balance sheet of the ultimate fate of ProL in INS
cells beginning from the zero chase time, we estimate that
of 100 newly synthesized molecules, ~50 molecules of
ProL are lost by secretion throughout a 20-h unstimulated
chase period, ~20 molecules are converted to mature L
and subsequently turn over intralysosomally, while ~30
molecules are still present as intracellular ProL, such that
when ~50% of insulin undergoes stimulus-dependent exocytosis during the last 4 h of the experiment, approximately half of these 30 molecules of labeled ProL are
released in parallel. For ProB in INS cells, the major difference appears to be that after a 20-h chase, ~50 molecules remain intracellularly as mature lysosomal cathepsin
B, whereas <1 molecule remains in secretory granules for
exocytosis. Mature All of these results point to the idea that in pancreatic
Active debate continues about different protein sorting
mechanisms involved in the formation of mature secretory
granules and the fidelity of sorting in different model systems. Our studies of the clonally derived INS cell line indicate that (a) secretory granules in the Prohydrolase sorting at the TGN is a complex process
based on the use of predominantly previously synthesized
MPRs for the sorting of newly synthesized lysosomal enzymes (Kornfeld and Mellman, 1989 Because prohydrolase sorting efficiency in the TGN of
different regulated secretory cells can vary, investigations
need to be extended to exocrine and other regulated
secretory cell types to determine the extent to which protein sorting during granule maturation is generally applicable or limited to selective systems. However, we note
that constitutive-like secretion has been observed in exocrine tissues (Arvan and Castle, 1987), whereas
secretory granules are apparent only in a subgroup
of cell types specialized for regulated secretion of certain
proteins that are stored at high concentration (Kelly, 1985
;
Burgess and Kelly, 1987
). In recent years, studies of specialized cell types have reported structural and functional
overlap between lysosomes and secretory granules (Taugner
and Hackenthal, 1988
; Bonifacino et al., 1989
; Burkhardt et
al., 1989
, 1990
), including the idea that a fraction of lysosomal enzymes/proenzymes may exist in the regulated secretory pathway of endocrine and exocrine tissues under
physiological conditions (Brands et al., 1982
; Docherty et
al., 1984
; Tooze et al., 1991
; Hirano et al., 1992
).
) or "zipper" them
(Kelly, 1985
; Kelly, 1991
; Tooze and Stinchcombe, 1992
)
into forming granules. Thus, it has been envisaged in sorting for entry models that at the time of entry into immature granules (IGs), regulated secretory proteins are either
bound to receptors (Moore et al., 1989
) or bound to other
regulated secretory proteins that are bound to receptors
(Palmer and Christie, 1992
; Pimplikar and Huttner, 1992
;
Leblond et al., 1993
; Yoo, 1993
; Natori and Huttner, 1996
;
Cool et al., 1997
), while nongranule secretory proteins remain soluble and are excluded from entry into IGs (Bauerfeind and Huttner, 1993
), being shunted to the constitutive
secretory pathway by default (Moore et al., 1983
; Kelly,
1985
). Alternatively, the "sorting by retention" hypothesis
argues that proteins need not enter IGs in an aggregated
state but may be soluble; subsequently, much of the condensation (insolubility) of regulated secretory proteins is
promoted within IGs (Garreau de Loubresse et al., 1994;
Gautier et al., 1994
; Kuliawat and Arvan, 1994
; Huang and Arvan, 1994
, 1995
). Condensation limits the ability of regulated secretory proteins to escape from maturing granules
by constitutive-like vesicle budding (Arvan and Castle,
1987
; Arvan and Chang, 1987
; vonZastrow and Castle, 1987
;
Arvan et al., 1991
; Grimes and Kelly, 1992
; Kuliawat and Arvan, 1992
), while other lumenal proteins may be removed by
these constitutive-like vesicles (Arvan and Castle, 1992
).
-cells of pancreatic islets, 99% of newly synthesized proinsulin may enter IGs (Rhodes and Halban, 1987
),
which are the major if not exclusive site of proinsulin endoproteolytic conversion to C-peptide and insulin (Orci et al.,
1985
, 1994; Rhodes et al., 1987
; Steiner et al., 1987
; Huang
and Arvan, 1994
; for review see Halban, 1994
). Under unstimulated conditions, the release of newly synthesized
C-peptide in molar excess of newly synthesized insulin can
be accounted for by the constitutive-like secretory pathway (Arvan et al., 1991
), which is hypothesized to be initiated by clathrin-coated vesicle budding from IGs (Kuliawat and Arvan, 1992
). Indeed, clathrin patches and buds
can be observed on IG membranes but are no longer
present on mature granules (Tooze and Tooze, 1986
; Orci
et al., 1987b
), while treatments that inhibit the loss of
clathrin from maturing granules (Orci et al., 1984a
) largely
block initiation of the constitutive-like secretory pathway (Kuliawat and Arvan, 1992
). Presumably, the immediate
target of these IG-derived clathrin-coated vesicles is endosomes, compartments from which some newly synthesized
proteins can be relocated to the cell surface (Fishman and
Fine, 1987
; Futter et al., 1995
).
; Mauxion et al.,
1996
), and further, the AP-1 adaptors can associate with
IGs (Dittie et al., 1996
). If, in regulated secretory cells,
MPRs can include the IG compartment as well as the
TGN in their trafficking itinerary, this could account for
the presence of lysosomal enzymes in secretory granules.
Specifically, it has been proposed that in large part, the
specialized lysosomal enzyme precursors of cytotoxic lymphocytes are targeted to secretory granules via MPRs
(Griffiths and Isaaz, 1993
). By contrast, we have reported
that in pancreatic islets treated with tunicamycin, the entry
of unglycosylated procathepsin L into IGs appears unimpaired, implying that MPRs facilitate a step other than
proenzyme entry into this compartment (Kuliawat and
Arvan, 1994
).
-cells account for the stimulated release of prohydrolases from pancreatic islets? Third, given reports of stimulated externalization of lumenal contents from
the endosomal system (Bomsel and Mostov, 1993
; Hansen
and Casanova, 1994
) and the fact that lysosomal proenzymes normally traffic through this system (Ludwig et al.,
1991
), do immature secretory granules really contain hydrolase precursors, or does stimulated proenzyme discharge derive from externalization of endosomal contents? Fourth,
could procathepsin L targeting to lysosomes in
-cells be
mediated largely by a non-MPR-dependent sorting mechanism? To explore these questions, we have compared the
targeting in
-cells of two close relatives in the papain superfamily of thiol proteases (Barrett and Kirschke, 1981
;
Takio et al., 1983
; Koga et al., 1990
) with distinct affinity for MPRs: procathepsin B (ProB) exhibits a high affinity
(Hanewinkel et al., 1987
), while procathepsin L (ProL) exhibits low-affinity MPR binding (Dong et al., 1989
; Dong
and Sahagian, 1990
; Lazzarino and Gabel, 1990
; Stearns
et al., 1990
). We report that from studies of the INS-1
-cell line and mouse islets, a significant fraction of both
proenzymes enters IGs, after which ProB is more efficiently removed from maturing granules. Moreover, a
small but definite decrease in the efficiency of ProB removal
from IGs is observed in the islets of transgenic mice lacking one of the two types of MPR, the cation-dependent MPR.
On the other hand, lysosomal proenzyme sorting in the
TGN and IGs is a dynamic process, underscored by new
observations that in islets isolated from male SpragueDawley rats, newly synthesized ProB can reach lysosomes
without appearing in large quantity in the stimulus-dependent secretory pathway. These data support a view of IGs
as a functional continuation of the TGN (Arvan and Castle,
1992
). Thus, to the degree that lysosomal enzyme precursors
enter IGs, the efficiency of their subsequent sorting appears
related to MPR recognition, leading to a clathrin-coated
vesicle escape route from maturing secretory granules.
Materials and Methods
) and
cathepsin L were graciously provided by Drs. J. Mort (Shriners Hospital,
Montreal, Canada) and G. Sahagian (Tufts University, Boston, MA), respectively. For immunoelectron microscopy, an mAb directed against a
proinsulin cleavage site (Orci et al., 1986
), a polyclonal antibody against
rat cathepsin B, and anticlathrin (Hille et al., 1992
) were obtained from
Drs. O Madsen (Hagedorn Research Institute, Copenhagen, Denmark),
A. Saluja (Beth Israel Hospital, Boston, MA), and F. Brodsky (University
of California, San Francisco), respectively. Guinea pig antiinsulin was from Linco (St. Louis, MO). Horseradish peroxidase-conjugated anti-rabbit serum (Cappel/Worthington) was used as a secondary for Western blotting (enhanced chemiluminescence; Amersham Corp., Arlington Heights,
IL). Collagenase P was from Boehringer Mannheim (Indianapolis, IN);
hypaque, human serum albumin, BSA, soybean trypsin inhibitor, other
protease inhibitors, tunicamycin, DTT, and iodoacetamide were from
Sigma Chemical Co. (St. Louis, MO); calf serum and antibiotic-antimycotic solution were from GIBCO-BRL (Gaithersburg, MD); [35S]methionine/cysteine (Expre35S35S) was from New England Nuclear (New
Bedford, MA); zysorbin-protein A was from Zymed Labs (So. San Francisco, CA).
). INS cells were washed twice with methionine-free, cysteinefree DME before pulse labeling for 30 min at 37°C in the same medium
containing ~100 µCi of [35S]methionine and cysteine per two million cells.
At the conclusion of the pulse labeling, the cells were washed and chased
in complete DME containing 1.67 mM glucose. For studies of nonglycosylated lysosomal enzymes, cells were pretreated with 20 µg/ml tunicamycin or mock-treated for 5 h. Rat islets (Arvan et al., 1991
) and mouse islets
(Kuliawat and Arvan, 1994
) were prepared, pulse labeled, and chased exactly as described previously. All labeling and chase media also contained
0.5 µg/ml human serum albumin (plus 0.005% soybean trypsin inhibitor
for islets). When comparing islets from adult male and female MPR-deficient mice (Ludwig et al., 1993
) vs. control males and females matched for
age and parentage (C57B/129), identical protocols were followed. All
rat islets were prepared from 275-g male Sprague-Dawleys supplied by
Charles River Labs, Inc. (Wilmington, MA). Granule exocytosis was stimulated in DME with 0.5 mg/ml RIA grade BSA plus a combination secretagogue including glucose (10 mM for INS cells, 22 mM for islets), 1 µM
phorbol 12-myristate 13-acetate, 1 mM isobutyl-methylxanthine, and 1 mM tolbutamide (and 10 mM leucine and glutamine for INS cells).
). The products of the two insulin genes expressed
in rodents were not distinguished from one another. The insulin gels were
fixed initially in 20% TCA (20 min), then in 12.5% TCA plus 50% methanol (15 min), then bathed in water (5 min), and finally incubated with 1 M
Na salicylate for 15 min. Dried gels were exposed to XAR film at
70°C.
Scanned x-ray films were finally analyzed using the ImageQuant program (Molecular Dynamics, Sunnyvale, CA).
). Multiple immunogold labeling was performed as described (Slot et al., 1991
), after
which the grids were analyzed in an electron microscope (model 1010;
JEOL U.S.A., Inc., Peabody, MA). Quantitation of mean gold particle density per granule was determined from secretory granules encountered at random by counting the number of gold particles representing either cathepsin B or cathepsin L in sections that were double immunolabeled for
proinsulin. Granule profiles that contained proinsulin label were marked
as IGs. By videoplanometer, mean surface area measured in the same sections was not significantly different for proinsulin-immunopositive and
proinsulin-immunonegative granules. In total, cathepsin B labeling was
counted in 45 IGs and 60 mature granules, while 55 IGs and 59 mature
granules were analyzed for cathepsin L.
Results
-cell line, INS-1
(Asfari et al., 1992
), which exhibits regulated exocytosis of
insulin in a nearly linear fashion during prolonged (4 h)
stimulation (Neerman-Arbez and Halban, 1993
). After a
30-min pulse labeling with 35S-amino acids, stimulated or
unstimulated secretions from labeled INS cells were sampled at different 90-min chase intervals during an 8.5-h time
course. At all chase times, secretagogue-induced exocytosis of newly synthesized insulin (or proinsulin) was obtained (Fig. 1 A, left). During the interval from 15-105 min
of chase, stimulus-dependent secretion of ProB could be
elicited (Fig. 1 B, left). By quantitative scanning densitometry, the stimulus-dependent release of ProB represented
17% of all immunoprecipitable B obtained at the 105-min
chase time and 37.3% of immunoprecipitable ProB obtained at this chase time, while stimulus-dependent secretion of immunoprecipitable insulin-containing peptides was
32% during the same interval. These data indicate that a
significant fraction of ProB entered the regulated secretory pathway. Of course, ProB delivery to lysosomes was
also ongoing during this chase period, as judged by processing in the cell lysates to the mature 31-kD cathepsin B. Indeed, at times >3 h of chase, labeled ProB was essentially absent from INS cell lysates (Fig. 1 B, right, and data
not shown), indicating that its arrival in lysosomes was complete. Consequently, in response to secretagogue addition after 3 h of chase, despite continued regulated exocytosis of labeled insulin (Fig. 1 A, left), INS cells did not
release any form of cathepsin B into the medium (Fig. 1 B,
left), indicating that neither ProB nor mature cathepsin B
remained to any significant extent in the secretory pathway.
Fig. 1.
Stimulus-dependent release
from INS cells of peptides immunoprecipitable with antiinsulin (A) or anti-
cathepsin B (B). The cells, pulse labeled as described in Materials and
Methods, were chased for up to 8.5 h.
During different 90-min chase intervals, either stimulated (+) or unstimulated () secretion was collected (left),
and the cells were then lysed. Equal
fractions of media and cells were immunoprecipitated using anti-ProB, and
one-tenth of these volumes were taken
for precipitation with antiinsulin. The
positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProB, and
mature cathepsin B (B) are shown.
Scanning densitometry of these gels
led to the numerical data described in
the text for this representative experiment (of three).
[View Larger Version of this Image (70K GIF file)]
;
Lazzarino and Gabel, 1990
). Because of the possibility of
rapid intracellular transport of ProL (Dong et al., 1989
;
Kane, 1993
) and long stimulation times required to elicit
granule exocytosis from INS cells (Neerman-Arbez and
Halban, 1993
), we initially screened for ProL secretion beginning 5 min after a 30-min pulse labeling (Fig. 2). As
shown during the first 2 h of chase, while proinsulin to insulin conversion was clearly ongoing (Fig. 2 A), secretagogue-induced secretion of ProL was also observed (Fig.
2 B). Evidently, like ProB, newly synthesized ProL could
also enter a stimulus-dependent secretory pathway in INS
cells. However, unlike ProB, at chase times up to 6 h, only
~20% of ProL ever reached lysosomes, as judged by conversion in the cell lysates to the bands comprising processed forms of mature L (Fig. 3). A 3-h secretagogue exposure elicited secretion of
50% of granule insulin from
INS cells, in agreement with previous reports (NeermanArbez and Halban, 1993). Importantly, the residual fraction of labeled ProL (Fig. 3, right) exhibited comparable
stimulated secretion (Fig. 3, left).
Fig. 2.
Stimulus-dependent release from INS cells of peptides
immunoprecipitable with antiinsulin (A) or anti-cathepsin L (B).
The cells were pulse labeled and chased as in Fig. 1, except that the stimulated (+) or unstimulated () medium collections began at 5 min of chase, were 60 min in duration, and were terminated at 2 h of chase. Only the medium is shown. While stimulated secretion of ProL is seen at all chase times, note the progression of
processing from proinsulin to insulin in the regulated secretory
pathway. Measurement of stimulus-dependent secretion during a
1-h period for L-containing peptides (~16%) was in a similar
range to that of insulin-containing peptides (~21%) in this experiment. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProL, and bands
comprising mature cathepsin L (Mr ~32,000 and ~27,000, respectively, large bracket) are shown.
[View Larger Version of this Image (59K GIF file)]
Fig. 3.
Inefficient arrival
of newly synthesized ProL in
lysosomes of INS cells. Cells
pulse labeled as in Fig. 1 were
chased to 3 h before a further
3-h collection of stimulated
(+) or unstimulated () secretion was obtained and the
cells analyzed by immunoprecipitation for lysosomal
arrival of cathepsin L. Stimulus-dependent secretion contained ~50% of all immunoprecipitable L-containing peptides found in the 3 h of collected
medium plus cell lysate, while insulin secretion as measured by
RIA averaged 53% during this period. The positions of ProL and
bands comprising mature cathepsin L (large bracket) are shown.
[View Larger Version of this Image (62K GIF file)]
Fig. 4.
A significant fraction of ProL in INS cells becomes entrapped in long-term storage in the regulated secretory pathway.
The stimulated (+) and unstimulated () secretions from pulselabeled INS cells were collected either during the first 4 h after labeling or after a chase of 20 h to first allow all newly synthesized ProL molecules to reach their final targets. At the conclusion of
each set, both media and cell lysates were analyzed by immunoprecipitation with anti-cathepsin L. Two exposures of the media,
differing fivefold, are shown in the upper two panels. Note the
disappearance of labeled forms of mature L at later chase times,
and the persistent stimulus-dependent secretion of the precursor.
The positions of ProL and bands comprising mature cathepsin L
(large bracket) are shown.
[View Larger Version of this Image (62K GIF file)]
), and this interaction occurs even for unglycosylated ProL synthesized after tunicamycin treatment (McIntyre and Erickson, 1993
; McIntyre et al., 1994
). For this reason, we examined the effects
of tunicamycin to block N-linked glycosylation and thereby
prevent formation of the mannose-6-phosphate (M6P)
moiety on ProL. It has previously been shown that such inhibition does not prevent the entry of ProL into the stimulus-dependent secretory pathway in mouse
-cells (Kuliawat and Arvan, 1994
); however, whether unglycosylated
ProL can escape from maturing granules has never previously been examined.
Fig. 5.
Stimulus-dependent release
from INS cells treated with tunicamycin.
Untreated INS cells (left) or those pretreated with tunicamycin (right) were
pulse labeled and chased for 3.5 h before
60-min collections of stimulated (+) or unstimulated () secretion, or the resulting
cell lysates were analyzed by immunoprecipitation with antiinsulin (A) or anti-
cathepsin L (B). For INS cells treated with
tunicamycin, samples were intentionally
double loaded to enhance the sensitivity of
detection of mature cathepsin L forms. Note that after tunicamycin treatment, the
amount of intracellular precursor increased disproportionately, as did the
amount of stimulus-dependent secretion
of ProL. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), glycosylated and unglycosylated ProL, and bands comprising glycosylated and unglycosylated mature cathepsin L (large brackets) are
shown.
[View Larger Version of this Image (66K GIF file)]
-Cells
) seemed consistent with
the hypothesis that a fraction of prohydrolases, unsorted
at the level of the TGN, may enter IGs and thereby acquire the property of regulated secretion. Subsequently,
ProB is efficiently removed from IGs, while most intragranular ProL fails to be removed. To further explore this
hypothesis, we examined ultrathin cryosections of mouse
pancreatic islet tissue to directly localize these antigens in
-cells by immunoelectron microscopy.
-cells, one theoretical concern was the
ability to discriminate between insulin-containing lysosomes
(Orci et al., 1984b
; Halban et al., 1987
) and true secretory
granules. In fact, lysosomes, tubular lysosomes, and crinophagic lysosomes were all unmistakably distinguishable
from secretory granules by immunoelectron microscopy,
as well as by conventional morphological criteria (Fig. 6,
A-C). Further, to reliably differentiate IGs from mature granules, we capitalized on the existence of a monoclonal
antibody to proinsulin, whose immunoreactivity is lost
upon processing to insulin (Orci et al., 1986
). Thus, when
examining
-cells of mouse islets, mature granules that
have little residual proinsulin remain unlabeled, while IGs
of different ages contain varying degrees of labeling (Fig.
7). When double or triple labeling with this mAb was performed along with anti-cathepsin B, cathepsin immunoreactivity was clearly evident in early granules (Figs. 7 and 8,
A and B). In addition, proinsulin-positive IGs not infrequently exhibited electron-dense coated membrane buds
similar to coated vesicles forming in the TGN. Using an antibody to clathrin, coated buds on both IGs and the TGN
were specifically decorated (Fig. 9). However, not only did
such buds appear absent from mature granules, but cathepsin B immunolabeling also appeared far less abundant over
mature granules (Figs. 7 and 8, A and B).
Fig. 6.
In -cells of mouse pancreatic islets, lysosomes are
morphologically distinct from secretory granules. (A) A typical
lysosome (L) juxtaposed next to a mature secretory granule
(star). The section was triple immunolabeled for C-peptide (5-nm
gold), insulin (10-nm gold) and cathepsin B (15-nm gold). (B) Tubular lysosomes (L) immunopositive for cathepsin B (10-nm
gold). (C) A crinophagic vacuole (L) juxtaposed next to a mature
secretory granule (star). Triple immunolabeling was as in A. Consistent with previous reports (Orci et al., 1984b
; Halban et al.,
1987
), C-peptide labeling is absent from lysosomes, although insulin labeling may be present. Bars, 200 nm.
[View Larger Version of this Image (109K GIF file)]
Fig. 7.
Ultrathin cryosection of the Golgi (G) region of a mouse islet -cell, triple immunolabeled for proinsulin (5-nm gold), cathepsin B (10-nm gold), and insulin (15-nm gold). Insulin label was found over all compartments of the secretory pathway. Granule profiles with the presence of proinsulin label were used to define IGs (I), while those with insulin labeling only and a variable-sized halo between the content and surrounding membrane were considered mature granules (M). A lysosome (L) is seen to be labeled exclusively for cathepsin B, although additional cathepsin B label can be seen in IGs. mito, mitochondrion. Bar, 200 nm.
[View Larger Version of this Image (109K GIF file)]
Fig. 8.
The occurrence of cathepsin B in IGs (i) of mouse islet -cells. Ultrathin cryosections were double immunolabeled for proinsulin and cathepsin B. Cathepsin B label (arrowheads) was clearly present over proinsulin-positive IGs (i), whereas mature granules (M) were almost devoid of label. (A) Proinsulin (5-nm gold); cathepsin B (10-nm gold). Coated buds (arrows) were often observed on IGs, especially in the Golgi (G) region. Immunoreactive cathepsins are occasionally found directly in the buds, but, similar to reports from
nonregulated secretory cells (Geuze et al., 1984
, 1985
; Ludwig et al., 1991
), immunogold detection of hydrolase precursors tends to be
impaired directly in the bud region. (B) Proinsulin (5-nm gold); cathepsin B (15-nm gold). L, lysosome. Bars, 200 nm.
[View Larger Version of this Image (176K GIF file)]
Fig. 9.
Coated buds that form on IGs contain clathrin. Ultrathin cryosections were immunolabeled with an antibody to
clathrin heavy chain. Labeling is present on numerous vesicular
profiles at the trans-side of the Golgi complex (G) and on a membrane bud of an IG (i), marked with arrows. Bar, 200 nm.
[View Larger Version of this Image (175K GIF file)]
-granules (identified with antiinsulin) were
strongly, albeit heterogeneously, immunolabeled for cathepsin L (Fig. 11, A and B). Moreover, in triple immunolabeling, mature
-granules that could be strongly labeled for
cathepsin L contained cathepsin B labeling at only low levels (Fig. 11 C). A quantitative analysis of
-cells immunolabeled with antiproinsulin was then performed to estimate the relative concentration of cathepsin B between
IGs and mature granules. The mean labeling density fell
an order of magnitude from 2.3 ± 0.35 (SEM) over IGs to
0.23 ± 0.06 over mature granules, and this decline was statistically significant (P < 0.005). In similar double labelings with anti-cathepsin L, the mean labeling density over
IGs (2.5 ± 0.25) was not significantly different from that over mature granules (2.5 ± 0.68). These data clearly indicate that in mouse islets, both proenzymes can enter
-cell
IGs, but ProL tends to remain to a much higher degree
than ProB in mature storage granules.
Fig. 10.
The occurence of cathepsin L in IGs and mature granules of mouse islet -cells. Ultrathin cryosections were double immunolabeled for proinsulin (5-nm gold) and cathepsin L (10-nm gold). (A) Overview of the Golgi (G) region. Cathepsin L labeling (small arrowheads) was clearly seen over proinsulin-positive IGs (i). The arrows point to coated buds on IGs. (B and C) Cathepsin L label was
abundantly observed over both immature (i) and mature granule profiles (M), although some granules were more extensively labeled
(large arrowheads) than others. mito, mitochondrion; P, plasmalemma. Bars, 200 nm.
[View Larger Version of this Image (168K GIF file)]
Fig. 11.
Immunolabeling of cathepsins L and B in mature granules of mouse islet -cells. (A and B) Ultrathin cryosections were immunolabeled for cathepsin L (10-nm gold) and insulin (15-nm gold), demonstrating the presence of cathepsin L labeling in mature
-granules. In B, two exocytotic profiles at the plasma membrane (P) are seen. (C) Triple labeling for insulin (5-nm gold), cathepsin L,
(10-nm gold), and cathepsin B (15-nm gold) demonstrates a mature granule immunopositive for cathepsin L and immunonegative for
cathepsin B (arrow). Occasional immunogold labeling of cathepsin B is indicated by arrowheads. Bars, 200 nm.
[View Larger Version of this Image (156K GIF file)]
). While M6Pdependent sorting of procathepsin D is thought to be mediated almost exclusively by cation-independent MPRs (CI-
MPRs), for most other M6P-containing ligands, CI-MPRs and cation-dependent MPRs (CD-MPRs) are used in combination, each to a greater or lesser degree (Ludwig et al.,
1994
; Kasper et al., 1996
). Because in INS cells and the
-cells of mouse islets, ProB, a high-affinity ligand for
MPRs, is efficiently sorted to lysosomes, while a significant fraction of ProL, a low-affinity ligand, remains in
secretory granules, the foregoing data imply that prohydrolase exit from maturing granules involves an MPRdependent mechanism. We therefore investigated the trafficking of ProB in the islets of adult mice with targeted disruption of both alleles encoding the CD-MPR (Ludwig et
al., 1993
) or age-matched controls from genetically similar
parentage.
; Kasper et al.,
1996
), it was not unexpected that in control experiments,
fibroblasts from the knockout mice yielded steady-state
levels of mature lysosomal cathepsin B that were only
slightly altered, and the sorting efficiency measured by
pulse-chase was reproducibly diminished by a modest (
10%) fraction (data not shown). Nevertheless, because
in normal mouse islets at later chase times, the stimulusdependent release of ProB from mature
-granules (i.e.,
"noise") is vanishingly small (Kuliawat and Arvan, 1994
),
it seemed plausible to detect changes in "signal" from missorted ProB stranded in mature
-granules of CD-MPR
knockout mice.
-granules (Fig. 12). In age-matched
control mice, ProB was barely detectable in the stimulusdependent secretion from mature
-granules, representing
only ~3% of all immunoprecipitable forms of cathepsin
B,2 whereas in the islets from CD-MPR-deficient mice,
this value was ~11%, i.e., increasing the missorted fraction of ProB nearly fourfold.
Fig. 12.
Stimulus-dependent exocytosis of mature
-granules releases an increased fraction of ProB from
the islets of transgenic MPRdeficient mice. Islets from
CD-MPR-deficient mice were
pulse labeled and chased
overnight in the absence of
secretagogues to allow the labeled proinsulin and ProB to
chase to their final destinations, before collecting sequential 30-min periods of unstimulated (
) and stimulated (+) secretion. See text for details. Secretion of labeled insulin and immunoprecipitable ProB were
analyzed by SDS-PAGE and fluorography.
[View Larger Version of this Image (46K GIF file)]
-Cells
) through
which newly synthesized proteins travel before their arrival in post-Golgi compartments (Kuliawat and Arvan,
1992
; Miller et al., 1992
; Rosa et al., 1992
; Xu and Shields,
1993
; Huang and Arvan, 1994
). Moreover, the potential
for clathrin-coated vesicle budding is shared by both the
TGN and IGs (Fig. 9). These facts raise an important question (considered further in the Discussion): Specifically, does the sorting of lysosomal hydrolase precursors from
TGN to endosomes always have the same efficiency in
regulated secretory cells generally, and in
-cells in particular? With this question in mind, we have been interested
to explore the extent of prohydrolase entry into
-cell IGs
from islets prepared from different animal models. We
therefore proceeded to examine the islets of normal male
Sprague-Dawley rats. Surprisingly, in spite of the brisk stimulated release of labeled proinsulin and insulin (Fig.
13 A), even at early chase times the presence of newly synthesized ProB in IGs (as detected by stimulus-dependent
secretion; Fig. 13 B, left) was obviously diminished from
that observed in INS cells (Fig. 1) or the islets of normal
mice (Kuliawat and Arvan, 1994
). Similar "negative" results
were obtained upon examination of a second high-affinity
MPR ligand, the ~75-kD
-glucuronidase precursor (data
not shown). By contrast, after tunicamycin treatment such that newly synthesized ProB was unglycosylated, ProB entrance into the regulated secretory pathway resumed (Fig.
13 B, right). Moreover, in the case of ProL, even without
tunicamycin treatment, stimulus-dependent secretion could
be detected in the islets of normal male Sprague Dawley
rats (Fig. 13 C). These data indicate that although lysosomal proenzymes may enter IGs in abundance, this entrance can vary considerably. Thus, IGs may functionally extend the TGN but do not serve as a mandatory intermediate in the trafficking of hydrolase precursors to lysosomes in all cases in regulated secretory cells.
Fig. 13.
Unstimulated and stimulated secretion of newly synthesized insulin (A), ProB (B), and ProL (C) from pancreatic
islets of normal male Sprague-Dawley rats. The islets, untreated
or pretreated with tunicamycin, were pulse labeled for 30 min.
During 30-90 min of chase, the islets were exposed either to unstimulated () or stimulated (+) conditions. Secretion of proinsulin + insulin was analyzed by immunoprecipitation as described (Arvan et al., 1991
). Immunoprecipitable ProB and ProL
were analyzed by SDS-PAGE and fluorography. After tunicamycin treatment, ProB was secreted as the unglycosylated form.
[View Larger Version of this Image (40K GIF file)]
Discussion
-cells.
These studies evolved from earlier work using tunicamycin treatment to investigate the trafficking of lysosomal
proenzymes, which led to the hypothesis that M6P recognition is likely to be used for prohydrolase escape from
maturing granules (Kuliawat and Arvan, 1994
). However,
because of theoretical concerns about the use of tunicamycin (see introduction), and because of recent reports of
regulated exocytosis that is not mediated by secretory
granules (Chavez et al., 1996
), herein we have taken several independent approaches, focusing especially on a comparative analysis of the trafficking of two homologous proenzymes, ProL and ProB, that exhibit low and high affinity for MPRs, respectively (Hanewinkel et al., 1987
; Dong
et al., 1989
; Dong and Sahagian, 1990
; Lazzarino and Gabel, 1990
; Stearns et al., 1990
).
). Importantly, using mouse islets, we have directly visualized
immunoreactive cathepsins L and B in
-cell IGs by immunoelectron microscopy (Figs. 8 and 10), and we have
further confirmed the significant persistence of immunolabeled cathepsin L in fully mature
-granules (Figs. 10 and
11). These data help to exclude the possibility that the regulated exocytosis of prohydrolases derives from alternative stimulus-dependent discharge pathways (Bomsel and
Mostov, 1993
; Hansen and Casanova, 1994
; Chavez et al.,
1996
)3. Instead, our findings strongly suggest that lysosomal proenzymes can enter IGs, and, while not directing
this entry, MPR association facilitates prohydrolase exit
from maturing
-granules.
-granules in isolated mouse islets also contain essentially no radiolabeled ProB (Kuliawat and
Arvan, 1994
). Thus, the biochemical and morphological
analyses presented in this report indicate that, because of a
low affinity for MPRs, the sorting efficiency of ProL during exit from maturing secretory granules is impaired approximately one order of magnitude compared to ProB.
Nevertheless, nearly all of the ProL that does successfully reach lysosomes requires carbohydrate-dependent sorting,4 and when this sorting is abrogated, even more ProL
accumulates in secretory granules (Fig. 5). Furthermore, in
the case of ProB, despite that CI-MPRs can direct the trafficking of most but not the entire fraction that becomes
available when both CD-MPR alleles are disrupted (these
results being consistent with independent reports [Kasper
et al., 1996
]), a distinctly increased fraction of ProB is
stranded in mature
-granules in the islets of transgenic mice that are normal except for the absence of CD-MPRs
(Fig. 12).
-cells, although lysosomal prohydrolases may be recognized by MPRs in the TGN and from there conveyed to
endosomes, should they fail to be sorted at the TGN, lysosomal proenzymes may then enter IGs. Nevertheless, this
does not automatically mean missorting because MPR recognition can still be used for escape from the regulated secretory pathway. Only the "doubly missorted" prohydrolases (i.e., those that fail to be sorted at both the TGN
and IGs) appear to become stranded as residents of mature secretory granules.
-cell can account
for the stimulated release of procathepsins previously observed in mouse islets and (b) by replicating the features
of lysosomal proenzyme trafficking, INS cells serve as a
valuable model system, in which biochemical analyses tend to be easier to execute than in primary islet preparations. However, because post-Golgi trafficking in professional secretory cells is physiologically regulated, no model
system can quantitatively represent all aspects of postGolgi sorting that at different times takes place under a diverse range of conditions.
). Aside from consideration of ligand affinities, the efficiency of TGN sorting
from moment to moment is based on dynamic changes in
the concentration of available ligands and MPRs fluxing
through the biosynthetic pathway. Notably, in
-cells, lysosomal biogenesis is modulated largely by the same factors that alternately stimulate either granule exocytosis or
crinophagy (Borg and Schnell, 1986
; Landstrom et al., 1988
,
1991; Schnell et al., 1988
; Borg et al., 1995
). Most importantly, protein flow rate through the secretory pathway,
which can be extraordinarily high in regulated secretory cells, influences the "lumenal dwell time" of the ligands
and receptors. This dwell time is much shorter in the TGN
than is the case for IGs (which can serve as a functional extension of TGN [Arvan and Castle, 1992
]). Thus, in certain
systems and under certain conditions, substantial fractions
of lysosomal precursors may escape the TGN and enter
IGs; by contrast, in other systems prohydrolase entry into
IGs can be greatly attenuated (Fig. 13 B). However, even
in the latter case, entry of lysosomal precursors into IGs
resumes under conditions when TGN sorting by MPRs is incomplete (Fig. 13, B and C).
; Arvan and Chang, 1987
; vonZastrow and Castle, 1987
; Arvan and Lee, 1991
) in
which there is evidence for progressive condensation of regulated secretory proteins in conjunction with progressive
loss of membrane by vesicular budding from IGs (Sesso,
A., B. Kachar, S.M. Carneiro, and I. Zylberman. 1990. J. Cell Biol. 111:312a), an increase in dry mass concentration
due to volume reduction during granule maturation (Wong
et al., 1991
), and, during size reduction, a decrease in the
granule membrane of the number of intramembranous
particles, which appear to be removed by the budding of
small vesicles heavily studded with such particles (Sesso et al.,
1980
). However, an important distinction may need to be
considered between granule maturation in endocrine and
exocrine cell types. Specifically, in endocrine cell types, as
granules mature, they become more acidic (Orci et al.,
1994
), and as the pH continues to drop towards pH 5, MPRs can no longer interact with lysosomal proenzymes.
Thus, even if MPRs remained available for clathrin-coated
vesicle budding late in granule maturation, they would not
efficiently remove lysosomal proenzymes, which would
then be missorted. Moreover, such acidity has the potential to autoactivate missorted hydrolases within granules,
leading to the possibility of digestion of intragranular contents (Neerman-Arbez and Halban, 1993
; Conlon et al.,
1995
). By contrast, mature exocrine granules do not appreciably acidify (Arvan et al., 1984
; Arvan et al., 1985
;
Arvan and Castle, 1986
; Orci et al., 1987a
). Thus, the only
factors that could cause lysosomal proenzyme missorting
to mature granules in these cells (Tooze et al., 1991
) would
be a limited availability of MPRs, the budding off of which
is thought to be a process kinetically limited to the period
of granule maturation (Arvan and Castle, 1992
). Clarification of these issues will clearly require further investigation.
Received for publication 30 September 1996 and in revised form 14 February 1997.
1. Abbreviations used in this paper: CD- and CI-MPR, cation-dependent and -independent mannose-6-phosphate receptors; IG, immature granules; M6P, mannose-6-phosphate; MPR, mannose-6-phosphate receptor; ProB and L, procathepsin B and L.This work was supported in part by National Institutes of Health grant DK48280 and a Research Grant from the American Diabetes Association to P. Arvan.