Sackler School of Graduate Biomedical Sciences, Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules.
Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.
Stress proteins, also known as heat shock proteins
(hsps)1, constitute several families of proteins that
were originally discovered because they were highly
inducible upon heat shock (Morimoto et al., 1994 Genetic and biochemical analyses indicate that organellar hsp70s are required for import of substrate proteins into
the ER (Vogel et al., 1990 When cultured cells are deprived of serum, rates of intracellular proteolysis increase (Amenta and Brocher, 1981 RNase A was originally studied as a substrate for the
hsc73-stimulated pathway of lysosomal proteolysis (Neff
et al., 1981 We have been able to reproduce the hsc73-stimulated
pathway of proteolysis using isolated lysosomes (Chiang
et al., 1989 Aniento et al. (1993) We now report colocalization of an hsp70 with the lysosomal marker protein lgp120 (Green et al., 1987 Cell Culture and Cell Fractionation
IMR-90 diploid human lung fibroblasts were maintained (Neff et al., 1981 Proteins, Antibodies, and Protein Assays
hsc73 purification from bovine brain cytosol was carried out according to
Welch and Feramisco (1985) One- and Two-dimensional Gel Electrophoresis
One-dimensional SDS-PAGE was carried out as previously described
(Maekawa et al., 1989 Immunoblotting
Proteins were electrotransferred onto either nitrocellulose (Schleicher & Schuell, Keene, NH) or Immobilon-P (Millipore Corp., Bedford, MA)
membranes. Immunoblotting with mAb 13D3 and mAb 7.10 was carried
out as described (Maekawa et al., 1989 After transfer of the two-dimensional gels to Immobilon-P, mAb 13D3
bound was visualized by incubation for 1 h with HRP-conjugated affinitypurified goat anti-mouse IgM, µ chain specific (Jackson ImmunoResearch Laboratories, Inc.) at a 1:10,000 dilution. The peroxidase was visualized by chemiluminescence using reagents from the ECL detection system
(Amersham Life Science, Inc., Arlington Heights, IL) and exposure to either X-OMAT AR film (Eastman Kodak Co., Rochester, NY) or Hyperfilm-ECL (Amersham Life Science, Inc.).
Immunoprecipitation
The mAb 13D3 or an irrelevant IgM was preincubated at 25°C with a mixture of short peptides to eliminate nonspecific binding. This mixture consisted of the dipeptides, MK, RE, and KF, and the hexapeptide, LTMRFA, each at 3 mg/ml. Purified hsc73 or PBP74 (1 µg) was incubated with
8 µg of mAb 13D3 at 37°C for 3 h followed by the addition of another 8 µg
of mAb 13D3 and an overnight incubation at 4°C. The samples were centrifuged for 15 min at 13,600 gmax at 25°C. Pellets were washed three times
with 2× PBS containing 0.2% Tween-20 and 0.02% NaN3, and then once
with PBS, containing 0.1% Tween-20 and 0.02% NaN3. The pellets were
then resuspended in gel loading buffer and run in 8% polyacrylamide gels.
Indirect Immunofluorescence and Confocal Microscopy
The cells grown on glass coverslips were washed five times with serumfree culture medium, and then fixed in
Trypsinization of Hsc73 and Ly-hsc73
Purified bovine brain hsc73 (12.5 µg/ml) was incubated with 0, 200, 400, or
800 µg/ml trypsin in a final vol of 100 µl in the presence or absence of 1%
Triton X-100 at 4°C for 20 min. Soybean trypsin inhibitor was added at
1.375-fold the concentration of trypsin, and lysosomal proteins were precipitated with 12% TCA containing 0.1% sodium deoxycholate. The resultant pellet was resuspended in gel loading buffer, and the pH was neutralized with 25% NH4OH.
Lysosomes from 2 × 106 cells were pelleted as described (Terlecky and
Dice, 1993 Antibody Endocytosis and Intracellular
Protein Degradation
Confluent IMR-90 fibroblasts in six-well plates were radiolabeled with 25 µCi/ml [3H]l-leucine (131 Ci/mmol; ICN Biomedicals, Inc., Irvine, CA)
for 2 d. Cell monolayers were washed four times with HBSS (GIBCO
BRL, Gaithersburg, MD) and incubated overnight at 37°C in 1 ml of fresh medium containing 10% newborn calf serum (NCS) with or without the
following: (a) 10 µg mAb 13D3, (b) 10 µg mAb P32, or (c) 10 µg mAb
13D3 plus 12 µg hsc73 (preincubated overnight at 4°C). Cell monolayers
were washed four times with HBSS and chased for 1 h with 6 ml of fresh
medium containing 10% NCS. Cell monolayers were then washed four
times with HBSS and cultured with fresh medium with or without 10%
NCS. Aliquots of media were taken over a time course, and protein degradation followed by measuring the radioactivity released into the medium
that was soluble in 3.5% phosphotungstate (PTA) and 5% HCl. Protein
degradation was calculated as described previously (Backer et al., 1983 Antibody Endocytosis and Degradation of
Endocytosed [3H]RNase A
Confluent fibroblasts in six-well plates were incubated overnight at 37°C
in medium plus 10% NCS containing 8 × 107 dpm of [3H]RNase A (2.96 × 104 dpm/pmol) and either 10 µg/ml of mAb 13D3, 100 µg/ml of mAb
13D3, 10 µg/ml of nonspecific IgM, 100 µg/ml of nonspecific IgM, or no
antibody. Cells were washed four times with HBSS containing 5% NCS
and three times with HBSS. Cells were returned to medium containing
10% NCS and, after 1 h, the medium was changed. Aliquots of media
were taken and the appearance of PTA/HCl-soluble radioactivity was determined.
Stability of Endocytosed Proteins
RNase A, mAb 13D3, and hsc73 were radiolabeled by reductive methylation using NaB3H4 as described (Backer et al., 1983 Counting of Radioactivity
Radioactivity was determined in a Beckman LS 1801 liquid scintillation
spectrophotometer using Ready Safe Scintillation Cocktail (Beckman Instruments, Inc., Fullerton, CA). Quenching was corrected by using an external radioactive standard.
Characterization of mAb 13D3
Previous studies have shown that mAb 13D3 recognizes
hsc73 but not the major heat-inducible hsp70 of human fibroblasts in immunoblots (Terlecky et al., 1992
Domanico et al. (1993) We next determined whether or not mAb 13D3 could
immunoprecipitate hsc73. Purified hsc73 and PBP74 were
directly immunoprecipitated with mAb 13D3 as described
in Materials and Methods. Immunoprecipitates and purified hsp70s were analyzed by immunoblots reacting either with mAb 13D3 (Fig. 1 D, lanes 1-4) or antiPBP74-1-16
(Fig. 1 D, lanes 5-8). mAb 13D3 immunoprecipitates
hsc73 (Fig. 1 D, lane 2) but does not immunoprecipitate
PBP74 (Fig. 1 D, lane 8). The higher molecular weight
band in lane 2 is due to altered mobility of a portion of the
hsc73 as a result of the high concentration of antibody required for the immunoprecipitation. Controls using a nonspecific IgM showed no detectable precipitation of hsc73 (data not shown). Furthermore, mAb 13D3 was able to
recognize native hsc73 in immunoblots performed at the
expected intralysosomal pH of 5.5 (data not shown).
Localization of ly-hcs73 by Confocal Microscopy
We examined and quantitated the lysosomal localization
of ly-hsc73 further by confocal microscopy. Double fluorescence staining of fibroblasts was carried out with mAb
13D3 and anti-lgp120 antibodies (Figs. 2 and 3). lgp120 is
the major glycoprotein in lysosomal membranes (Green
et al., 1987 To quantitate Fig. 2 and similar images, we scored 1,000 immunofluorescent vesicles. More than 90% of the lysosomes contained ly-hsc73, and >80% of the vesicular hsp70
was lysosomal. However, some lysosomes did not contain
detectable levels of ly-hsc73, visible as a pure red signal. In
addition, some nonlysosomal vesicular structures contain
an immunoreactive hsp70, visible as a pure green signal.
These organelles may be clathrin-coated vesicles and/or
peroxisomes since hsc73 is known to associate with these structures (Rothman and Schmidt, 1986; Walton et al., 1994 Fig. 3 compares cells grown in the presence (Fig. 3 B)
and absence (Fig. 3 A) of serum using a thick optical section (4 µm) that includes cytoplasm containing lysosomes
above the nucleus. This analysis shows a remarkable
change in the lysosomal morphology in response to serum
withdrawal. Lysosomes in serum-deprived fibroblasts appear to fuse, adopting a tubular shape (defined as structures with a length greater than four times their diameter).
These tubular lysosomes were not evident using the thin
optical section (Fig. 2) probably because the tubules repeatedly traverse the optical section. Tubular lysosomes
have been previously described in activated macrophages
(Swanton et al., 1987; Heuser, 1989 The Majority of ly-hsc73 Is in the Lumen
We characterized the amount of trypsin required to completely digest purified bovine brain hsc73 in 20 min at 4°C.
Fig. 4 A is an immunoblot using mAb 13D3 that shows
that 1% Triton X-100 does not alter the ability of mAb
13D3 to recognize hsc73 (Fig. 4 A, lanes 1 and 2), and that
200 µg/ml of trypsin is sufficient to completely digest hsc73
in the absence (lane 3) but not in the presence (lane 4) of
1% Triton X-100. To digest all of the hsc73 in the presence
of 1% Triton X-100, 800 µg/ml of trypsin was required
(Fig. 4 A, lane 9). The presence of 1% Triton X-100 did
not affect trypsin's activity against radiolabeled RNase A or BSA (data not shown), suggesting that the stabilization
was due to the detergent interacting with hsc73.
When isolated lysosomes were incubated with 800 µg/ml
trypsin and analyzed by immunoblotting with mAb 13D3,
only 25% of the signal was lost (Fig. 4 B, lanes 1 and 2).
However, when the lysosomes were permeabilized with
1% Triton X-100 before the incubation with trypsin, lyhsc73 was completely digested (Fig. 4 B, lane 3).
This luminal localization of the majority of ly-hsc73 is
further supported by our two-dimensional electrophoresis
analyses followed by immunoblotting with mAb 13D3.
Fig. 5 A shows the multiple isoforms of fibroblast cytosolic
hsc73. Fig. 5 B represents mixed ly-hsc73 and bovine brain
hsc73 purified as described previously (Welsh and Feramisco, 1985). Fibroblast cytosol and bovine brain cytosol
showed similar isoforms of hsc73 (data not shown). Fig. 5 C shows that the isoelectric point of the ly-hsc73 corresponds to the most acidic isoform present in the cytosol. A
longer exposure of the immunoblot allowed us to visualize
the presence of a small amount of the most abundant cytoplasmic hsc73 isoform (data not shown). This hsc73 isoform was localized to the lysosomal membrane (Fig. 5 D).
The cause of these charge variants is not known, but such
isoforms have also been reported for other cell types (Watowich and Morimoto, 1988
Role of ly-hsc73 in the hsc73-Stimulated Lysosomal
Proteolytic Pathway
We studied the possible role of ly-hsc73 in the selective lysosomal protein degradation pathway during serum withdrawal by attempting to block the ly-hsc73 with endocytosed mAb 13D3. To determine how much mAb 13D3 would
be needed to neutralize ly-hsc73, we radiolabeled mAb 13D3
with NaB3H4 and determined the amount of [3H]mAb 13D3
that could be endocytosed by fibroblasts and its half-life after endocytosis. These studies indicated that mAb 13D3
was internalized by absorptive endocytosis at 12 times the
rate of [3H]sucrose, a fluid-phase marker (Gurley and Dice,
1988 Chiang and Dice (1988) In contrast, the enhanced protein degradation in response to serum withdrawal (Fig. 6 A) was blocked when
the mAb was endocytosed alone (Fig. 6 B). Endocytosis of
mAb 13D3 previously neutralized with hsc73 (Fig. 6 C), or
the control antibody (P32; Fig. 6 D), had little effect. These
results were obtained in two additional experiments except that the slightly slower protein degradation observed
for the P32-treated cells (Fig. 6 D) was not a consistent finding.
To determine whether endocytosis of mAb 13D3 inhibited degradation of proteins once they had entered lysosomes, we incubated fibroblasts with [3H]RNase A as a
substrate for degradation together with mAb 13D3 or
mAb P32. Fig. 7 shows that mAb 13D3 had no effect on lysosomal degradation of endocytosed [3H]RNase A even at
a 10-fold higher concentration than that which inhibits the
enhanced intracellular protein degradation during serum
withdrawal.
The ly-hsc73 appears to be cytosolic hsc73 that enters lysosomes through macroautophagy or other processes and
is slightly modified to become more acidic. If ly-hsc73 is
hsc73, we reasoned that hsc73 might be particularly resistant to intralysosomal digestion. We measured the degradation rates of RNase A, a mixture of cytosolic proteins,
and two different preparations of hsc73 after endocytosis (Fig. 8). RNase A was degraded with a half-life of 47 h, while the half-lives of cytosolic proteins were heterogeneous in
the 20-60-h range (data not shown). Radiolabeled hsc73 that
was repurified by ATP-affinity chromatography before endocytosis was remarkably stable (t1/2 = 170 h). Degradation
of total reductively methylated hsc73, most of which could
not bind to ATP, was biphasic with rapid (t1/2 = 5.5 h) and
longer-lived (t1/2 = 26 h) components. These results are
consistent with most of the [3H]hsc73 being denatured by
the reductive methylation and short-lived after endocytosis, while the native [3H]hsc73 is very stable within lysosomes.
The experiments described here were aimed at further
characterizing ly-hsc73 and directly testing the role(s) of
ly-hsc73 in the hsc73-stimulated pathway of lysosomal proteolysis. We identified mAb 13D3 as a reagent that recognizes both the denatured and native forms of hsc73 without
cross-reacting with other known hsp70 family members
(Fig. 1).
We analyzed the subcellular distribution of ly-hsc73 in
methanol-fixed cells by laser scanning confocal microscopy (Figs. 2 and 3). ly-hsp73 is localized to most, but not
all, lysosomes; to a lesser extent, ly-hsc73, hsc73, or another
hsp70 that is recognized by mAb 13D3 is also associated
with other organelles.
Images generated using a thick optical section unexpectedly showed that lysosomes in fibroblasts have the capacity to fuse into a tubular network when cells are serum deprived. Tubular lysosomes have been described before in
other cell types (Novikoff and Shin, 1978 The morphological change in lysosomes could also be
unrelated to their degradative capacity. For example, certain organelles vesiculate before cell division presumably
to allow efficient partitioning into the two daughter cells
(Birky, 1983 Trypsin treatment of purified lysosomes (Fig. 4) indicated that ~25% of the lysosomal hsc73 is membrane
bound, while 75% is within the lysosomal lumen. These results are consistent with immunogold EM analysis of lyhsc73 distribution in rat liver lysosomes (Cuervo et al., 1995 Our two-dimensional gel analyses of hsc73 and ly-hsc73
(Fig. 5) indicate that most of the ly-hsc73 corresponds to the
most acidic isoform of cytosolic hsc73. Whether this isoform is preferentially taken up by lysosomes or whether
other isoforms are converted within lysosomes to more acidic
molecules remains to be established. A minor ly-hsc73 is
also of the same isoelectric point as the major cytosolic
hsc73 species and is confined to the lysosomal membrane
(Fig. 5 D). Additional experiments using purified rat liver
lysosomes indicate that cytosolic hsc73 can be internalized by lysosomes and is functional in stimulating the uptake of
substrate proteins (Cuervo et al., 1997 Hsc73 may enter lysosomes by a variety of mechanisms
including the hsc73-mediated pathway (Cuervo et al.,
1997 Our preliminary experiments concerning the endocytic
uptake and degradation of mAb 13D3 indicated that we
could deliver excess mAb 13D3 to lysosomes and that
mAb 13D3 could recognize hsc73 under the acidic conditions within lysosomes. The fact that the endocytosed
mAb 13D3 completely blocked the enhanced proteolysis in response to serum deprivation (Fig. 6) shows that the
endocytosed mAb 13D3 interfered with the function of lyhsc73.
An alternative interpretation of these results is that the
endocytosed mAb 13D3 somehow leaked from endosomes/
lysosomes into the cytosol and blocked the selective lysosomal degradation pathway by neutralizing cytosolic
hsc73. This possibility seems unlikely since, by indirect immunofluorescence in formaldehyde-fixed fibroblasts, we
were able to localize the majority of endocytosed mAb
13D3 to lysosomes (data not shown). In addition, Okada
and Rechsteiner (1982) There are several ways in which ly-hsc73 might be required for the hsc73-stimulated pathway of lysosomal proteolysis. ly-hsp73 might be required for the import of substrate proteins by mechanisms similar to those previously
proposed for other hsp70 family members in mitochondria
(Kang et al., 1990 These results together with previously defined features
of the hsc73-stimulated pathway of lysosomal proteolysis
lead us to the working model shown in Fig. 9. Cytosolic
hsc73, acting as a molecular chaperone, recognizes specific
sequences (KFERQ and related peptides) in proteins that
are targeted for lysosomal degradation in response to serum withdrawal. These sequences may be exposed by
changes in the conformation of the substrate proteins or
by alterations in protein-protein interactions. Alternatively, cytosolic hsc73 may be altered by serum withdrawal
in some way so that it now acts in the lysosomal degradative pathway rather than in organelle synthetic pathways
or in the uncoating of clathrin-coated vesicles.
The substrate protein is then recognized by the lysosomal membrane receptor protein lgp96 (Cuervo and Dice,
1996 The translocation of the substrate protein across the lysosomal membrane may require ly-hsc73, in an analogous
way to the roles of other intraorganellar hsp70s in the
complete import of proteins into mitochondria and the lumen of the ER (Kang et al., 1990 Two major models have been proposed to explain involvement of intraorganellar hsp70s in the protein translocation process across membranes. The Brownian ratchet
model predicts that Brownian motion drives the translocation of a polypeptide across membranes, and that luminal
hsp70s act simply by binding to their polypeptide substrates,
conferring vectorial transport (Simon et al., 1992). In addition, several heat shock proteins are constitutively expressed or are regulated by factors other than stress. Heat shock proteins of 70 kD (hsp70s) have been the subject of
considerable study (Craig et al., 1994
). These molecular
chaperones are localized to different cellular compartments where protein folding has to be achieved, altered, or
stabilized (Hendrick and Hartl, 1993
; Hartl, 1996
). One of
the members of this family of proteins, the constitutively
expressed heat shock cognate (hsc) protein of 73 kD
(hsc73), has been implicated in the mediation of clathrin
uncoating from endosomes (Rothman and Schmid, 1986
);
regulation of cytoskeletal interactions (Green and Liem,
1989
); transient association with nascent polypeptides (Beckman et al., 1990
); and facilitation of the import of polypeptides into mitochondria (Deshaies et al., 1988
; Murakami
et al., 1988
; Sheffield et al., 1990
), the ER (Chirico et al.,
1988
; Deshaies et al., 1988
), the nucleus (Shi and Thomas, 1992
), and peroxisomes (Walton et al., 1994
).
; Nicchitta and Blobel, 1993
; Panzner et al., 1995
) and mitochondria (Kang et al., 1990
; Rassow et al., 1994
). In these cases, a distinct hsp70 family member resides within the ER (mammalian glucose-regulated
protein of 78 kD [grp78]; yeast karyogamy gene 2 product)
and mitochondria (mammalian glucose-regulated protein
of 75 kD; yeast stress 70 subgroup C gene product).
;
Hendil et al., 1990
). Our laboratory has previously reported that serum deprivation of confluent human fibroblasts activates a selective pathway of lysosomal proteolysis
(Chiang and Dice, 1988
) and that cytosolic hsc73 stimulates this pathway of protein degradation (Chiang et al.,
1989
). This selective pathway of lysosomal proteolysis, which
we will hereafter refer to as the hsc73-stimulated pathway, is also activated in liver, kidney, and heart of starved rats (Chiang and Dice, 1988
; Wing et al., 1991
; Cuervo et al.,
1995
).
; Backer et al., 1983
). Residues 7-11 of RNase A,
KFERQ, are required for entry of RNase A into this
proteolytic pathway (Dice et al., 1986
). Furthermore, the
KFERQ pentapeptide is an important element in the binding of RNase A and RNase S-peptide (amino acids 1-20 of
RNase A) by hsc73 (Terlecky et al., 1992
). Approximately
30% of cytosolic proteins contain peptide sequences immunologically related to KFERQ, and such proteins are
also targeted to the hsc73-stimulated pathway of lysosomal proteolysis (Chiang and Dice, 1988
; Wing et al., 1991
).
; Terlecky et al., 1992
; Terlecky and Dice, 1993
;
Cuervo et al., 1994
, 1995
). Uptake of substrate proteins is
saturable, and specific binding to a lysosomal membrane
protein occurs before uptake. Lysosomal uptake and degradation of substrate polypeptides is stimulated by hsc73
and Mg2+-ATP, and it is activated by withdrawal of serum
from cells before isolation of lysosomes. This pathway of
proteolysis is inhibited by low temperatures, NH4Cl, and
leupeptin.
have shown that glyceraldehyde-3phosphate dehydrogenase is selectively taken up by rat
liver lysosomes in vitro. The process appears to be similar
to that for RNase A by fibroblast lysosomes since it is also
stimulated by Mg2+-ATP and hsc73. Furthermore, uptake
of glyceraldehyde-3-phosphate dehydrogenase can be competed with RNase A and vice versa (Cuervo et al., 1994
).
The pathway in rat liver lysosomes is activated by prolonged starvation (Cuervo et al., 1995
). Our laboratory has
recently identified the lysosomal glycoprotein (lgp) of 96 kD (lgp96) as the lysosomal receptor for this proteolytic
pathway (Cuervo and Dice, 1996
).
). The lysosomal hsp70 (ly-hsc73) appears to be primarily within the
lysosomal lumen. An mAb that recognizes hsc73 and lyhsc73 when delivered to lysosomes by endocytosis completely blocks this pathway of proteolysis. Thus, ly-hsc73
appears to play a critical role in the operation of this proteolytic pathway.
Materials and Methods
;
Terlecky and Dice, 1993
), and cells were fractionated into organelles and
cytosol (Chiang and Dice, 1988
) as previously described. The membrane
fraction was the pellet from the centrifugation at 100,000 g for 1 h, and the
supernatant from this centrifugation contained cytosolic proteins. Lysosomes were purified from the postnuclear supernatant as previously reported (Terlecky and Dice, 1993
). For immunolocalization, IMR-90 human diploid fibroblasts were cultured at low density in six-well plates
(Costar, Cambridge, MA) on glass coverslips. Half of the plates were serum deprived for 18 h, and the rest remained in serum-supplemented culture medium before fixation.
. The peptide binding protein (PBP) of 74 kD
(PBP74) was a gift from Drs. Diane DeNagel and Susan Pierce (Northwestern University, Evanston, IL). The stress 70 subgroup A1 protein
(SSA1p) was a gift of Drs. Bruce Koch and Randy Schekman (University
of California, Berkeley). Affinity-purified mouse mAb 13D3 against hsc73
(Maekawa et al., 1989
) was kindly provided by Joseph Chandler (Maine
Biotechnology Services Inc., Portland, ME). A polyclonal rabbit antibody
directed against rat lgp120 was a generous gift of Dr. Ira Mellman (Yale University, New Haven, CT). A polyclonal rabbit antibody raised against
amino acids 1-16 of PBP74 (antiPBP74-1-16; Domanico et al., 1993
) was
kindly provided by Drs. Diane DeNagel and Susan Pierce. Mouse mAb
P32 was a gift from Dr. Henry Wortis (Tufts University, Boston, MA).
This IgM recognizes
-1,3-dextran (Schilling et al., 1980
). Mouse mAb
7.10 was purchased from Affinity Bioreagents (Neshanic Station, NJ). Fluorescein-labeled secondary anti-mouse IgM, µ chain specific, and Texas
red-labeled anti-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Protein determinations
were performed using either the BioRad protein assay reagent (Bio Rad
Laboratories, Hercules, CA) or the Lowry assay (Lowry et al., 1951
).
; Terlecky et al., 1992
). Two-dimensional electrophoresis was performed essentially as described (O'Farrell, 1975
). IEF with pH
5.0-6.0 ampholines (Sigma Chemical Co., St. Louis, MO) was carried out
in 20-cm-long gels at 1.5 kV for 18 h.
; Terlecky et al., 1992
). Immunoblotting with antiPBP74-1-16 was performed either at 37°C for 3 h or overnight at 4°C at a 1:300 dilution.
20°C methanol for 1 min and
stored at 4°C in PBS containing 0.02% NaN3. For fluorescent double labeling of ly-hsc73 and lgp120, all incubations and washes were at 25°C.
Primary antibodies were diluted 1:50, and secondary antibodies were diluted 1:100 in PBS containing 0.1% BSA and 0.02% NaN3. Cells were incubated for 1 h with a mixture of primary antibodies, washed 10 times in
PBS, and incubated for 1 h with each of the corresponding secondary antibodies. Coverslips were mounted in mounting media containing an antibleaching agent (Kirkegaard & Perry Laboratories, Inc., Gaithersburg,
MD). Images were collected on a laser scanning confocal microscope, either an MRC 600 (Bio Rad Laboratories) or an ODYSSEY XL (NORAN
Instruments, Middleton, WI). The image analysis software used was INTERVISION (NORAN Instruments). Pictures were taken with Kodachrome 64 and TMAX400 film (Eastman Kodak Co.). Fig. 3 C was generated using Adobe Photoshop 3.0 software (Adobe Systems Inc., Mountain
View, CA). No fluorescence was associated with cells after incubation with secondary antibodies alone (data not shown).
Fig. 3.
Distribution of ly-hsc73 and
lgp120 in serum-deprived and serumsupplemented fibroblasts using a thick
optical section. Methanol-fixed fibroblasts were stained with primary antibodies to hsc73 (green) and lgp120
(red) as described in the legend to Fig.
2. The merged color images using an
optical section of 4 µm show: (A) serum-deprived fibroblast and (B) serum-supplemented fibroblast. Note
that lysosomes appear to fuse, forming
a tubular network when cells are serum deprived (A). Bar, 10 µm.
[View Larger Version of this Image (39K GIF file)]
Fig. 2.
Distribution of ly-hsc73 and lgp120 by confocal microscopy using a narrow optical section. Fibroblasts were serum deprived overnight, methanol fixed, and then incubated with mAb
13D3 and anti-lgp120 simultaneously followed by Texas red- and
fluorescein-conjugated second antibodies to reveal ly-hsc73 (green)
and lgp120 (red). The thickness of the optical section analyzed
was 0.09 µm. Colocalization of both proteins registers as yellow/
orange. (Insets) Left panel, fluorescein channel; right panel,
Texas red channel. Bar, 10 µm.
[View Larger Version of this Image (45K GIF file)]
), resuspended in a final vol of 100 µl of PBS with or without
800 µg/ml trypsin and 1% Triton X-100, and incubated at 4°C. After 20 min, soybean trypsin inhibitor (1.1 mg) was added and the samples were
treated as described above.
)
and plotted as the exponential loss of radioactivity associated with the
monolayer at increasing times. The calculated best fit lines and the halflives were obtained by Fig.P software (Biosoft, Cambridge, UK).
). Confluent cultures of
IMR-90 fibroblasts were labeled for 2 d with 12 µCi/ml of [35S]l-methionine
(422 Ci/mmol; ICN Biomedicals, Inc.), and cytosolic proteins were prepared. Radiolabeled proteins were added to cell monolayers in six-well
plates in medium containing 10% NCS. The amount of radioactivity
added per well and the specific radioactivities of the proteins were as follows: mAb 13D3: 1.75 × 106 dpm, 5 × 106 dpm/nmol; RNase A: 3 × 107
dpm, 2.4 × 105 dpm/nmol; hsc73: 2.5 × 107 dpm, 1.4 × 108 dpm/nmol; repurified hsc73: 5 × 105 dpm, 1.4 × 108 dpm/nmol; cytosolic proteins: 4 × 106 dpm, 2.1 × 104 dpm/nmol (assuming an average molecular mass of
40 kD). After an overnight incubation, cells were washed four times with
HBSS containing 5% NCS and three times with HBSS alone. Cells were chased in medium containing 10% NCS for 6 h, and the medium was then
changed. Monolayers were washed and harvested in PBS containing 1%
Triton X-100, and the PTA/HCl-precipitable radioactivity was determined.
Results
). mAb
13D3 also recognized hsc73, or another as yet unidentified
hsp70, in a cellular membrane fraction (see Materials and
Methods; Fig. 1 A, lane 1) but did not recognize what we
presume to be grp78 (Fig. 1 A, lane 2, upper band). Both
hsc73 and grp78 could be visualized by mAb 7.10, which is
known to recognize several members of the hsp70 family
(Kurtz et al., 1986
; Fig. 1 A, lane 2). In addition, mAb
13D3 did not recognize the yeast hsp70, SSA1p (Fig. 1 B,
lane 1), which is recognized by mAb 7.10 (Fig. 1 B, lane 2).
Fig. 1.
Immunoblot and immunoprecipitation analysis of mAb
13D3 specificity. 25 µg of fibroblast membranes (A), 1.5 µg of
SSA1p (B), or 4.5 µg of PBP74 (C) were separated by SDSPAGE and transferred to nitrocellulose membranes. Lanes were
probed with mAb 13D3, mAb 7.10, or antiPBP74-1-16 as indicated. Immunoprecipitations (D) were with mAb 13D3 and hsc73
or PBP74. (Lanes 1-8) Immunoblots of the purified proteins and
the immunoprecipitates. (Lanes 1 and 5) 0.2 µg hsc73; (lanes 2 and 6) hsc73 immunoprecipitated with mAb 13D3; (lanes 3 and
7) 0.2 µg PBP74; (lanes 4 and 8) PBP74 incubated with mAb
13D3. Lanes were probed with mAb 13D3 or antiPBP74-1-16 as
indicated.
[View Larger Version of this Image (26K GIF file)]
identified a new member of the
hsp70 family, PBP74, and showed that it was localized to
mitochondria (Dahlseid et al., 1994
). Fig. 1 C shows an immunoblot analysis of purified PBP74. A wide single lane of
an SDS-PAGE gel was transferred to a membrane, the
membrane was cut in thirds longitudinally, and each strip
was incubated with a different antibody. Lane 1 was incubated with antiPBP74-1-16, lane 2 with mAb 13D3, and
lane 3 with mAb 7.10. AntiPBP74-1-16 recognized purified
PBP74, but neither mAb 13D3 nor mAb 7.10 recognized
PBP74 (Fig. 1 C, lanes 2 and 3, respectively).
). As previously described, hsc73 is an abundant
cytosolic protein (Giebel et al., 1988
). To avoid the interference of cytosolic hsc73 during immunolabeling with mAb 13D3, cells were fixed in cold methanol, which did
not fix the cytosolic fraction of hsc73 and allowed us to examine organelle-associated hsp70s that are recognized by
mAb 13D3. The mAb 13D3 (green signal) largely colocalized with the anti-lgp120 antibodies (red signal). A thin
optical section (0.09 µm) shows that both proteins are confined to the cytoplasmic compartment (Fig. 2). Colocalization in the merged images (Figs. 2 and 3) registers as orange/yellow.
).
; Knapp and Swanson, 1990
), smooth muscle cells (Robinson et al., 1986
), endothelial cells (Kuijpers et al., 1994
), and hepatocytes (Novikoff and Shin, 1978
). We scored 1,600 immunofluorescent vesicular structures or equivalent areas within tubules
in cells deprived of serum. Once again, >90% of the lysosomes contained ly-hsc73, and >80% of the vesicular or
tubular hsp70 was lysosomal.
Fig. 4.
Protease digestion of purified hsc73 and ly-hsc73. Purified bovine brain hsc73 (A) and lysosomal proteins (B) were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with mAb 13D3. hsc73 (A) or lysosomes purified from
serum-deprived fibroblasts (B) were treated with buffer alone,
buffer plus trypsin, or buffer plus trypsin and 1% Triton X-100 as
indicated and described in Materials and Methods.
[View Larger Version of this Image (21K GIF file)]
; Bhattacharyya et al., 1995
).
Fig. 5.
Two-dimensional gel electrophoresis of hsc73 and lyhsc73. Fibroblast cytosol (150 µg protein; A), a mixture of fibroblast lysosomes (200 µg protein) and purified bovine brain hsc73
(1 µg protein; B), fibroblast lysosomes (200 µg protein), and fibroblast lysosomal membranes (100 µg protein; C) were separated as described in Materials and Methods. The membranes
were immunoblotted with mAb 13D3 and developed with the
ECL system. (Arrows) hsc73.
[View Larger Version of this Image (39K GIF file)]
), and it had a half-life within lysosomes of 45 h (data
not shown). The previous quantitation of the amount of lyhsc73 (Terlecky and Dice, 1993
) allowed us to calculate that
overnight endocytosis of 10 µg/ml of mAb 13D3 should result in an eightfold molar excess of mAb 13D3 over lyhsc73.
showed that the enhanced degradation of bulk cellular proteins within confluent fibroblasts in response to serum withdrawal was entirely due to
activation of the hsc73-stimulated pathway of lysosomal
proteolysis. Therefore, we radiolabeled confluent cultures
of fibroblasts for 2 d with [3H]leucine. These fibroblasts
were then incubated overnight in media containing 10%
NCS and either no antibody, mAb 13D3, mAb 13D3 preincubated with hsc73, or an irrelevant IgM, P32. Protein degradation during the overnight endocytosis period was
the same for all groups of cells (data not shown). This result makes it unlikely that endocytosis of any of the antibodies was generally toxic to cells.
Fig. 6.
Effect of mAb 13D3 on enhanced degradation of cellular proteins
in response to serum deprivation. Fibroblasts were labeled with [3H]leucine for 2 d, and then incubated overnight in medium containing 10% NCS
and either no addition (NONE), or
mAb 13D3 (13D3), mAb 13D3 preincubated with hsc73 (13D3/HSC73), or
mAb p32 (P32). The cells were chased
with fresh media containing 10% NCS
for 1 h, and then changed to serumsupplemented (solid lines) or serumdeprived (dotted lines) media. Acidsoluble radioactivity in the media was followed as a measure of proteolysis.
Results shown are the mean for n = 6 (NONE), or n = 4 (13D3, 13D3/
HSC73, and P32). The half-lives (in h)
in the presence and absence of serum,
respectively, are: (A) 80 and 52; (B) 81 and 84; (C) 100 and 74; and (D) 120 and 77.
[View Larger Version of this Image (21K GIF file)]
Fig. 7.
Effect of endocytosed antibodies on intralysosomal
degradation of endocytosed [3H]RNase A. Fibroblasts were incubated overnight in media containing 10% NCS and [3H]RNase A
alone (filled circles) or in combination with 10 µg/ml mAb 13D3
(filled squares), 100 µg/ml mAb 13D3 (triangles), 10 µg/ml of an
irrelevant IgM (P32; open circles), or 100 µg/ml of P32 (open
squares), and then chased 1 h with fresh media containing 10%
NCS. After the chase, fresh media containing 10% NCS was
added to each well, and acid-soluble radioactivity in the media
was determined at the indicated times. Results shown are the
mean for n = 4.
[View Larger Version of this Image (20K GIF file)]
Fig. 8.
Stability of endocytosed proteins. Fibroblasts were
incubated overnight in media containing: 35S-labeled cytosolic
proteins from IMR-90 fibroblasts (cytosol), [3H]RNase A, total
[3H]hsc73 (HSC73), or [3H]hsc73 that had been repurified by
ATP-affinity chromatography (HSC73*). Cultures were chased
for 6 h in media containing 10% NCS, and then degradation was
followed by measuring acid-soluble radioactivity appearing in the
medium. Results are the mean for n = 3-6.
[View Larger Version of this Image (17K GIF file)]
Discussion
; Robinson et al.,
1986
; Swanson et al., 1987
; Heuser, 1989
; Knapp and Swanson, 1990
; Kuijpers et al., 1994
) but not, to our knowledge,
in fibroblasts. It is possible that fusion of lysosomes favors
a more efficient degradation process by correcting heterogeneities among separate lysosomal vesicles. For example, if certain lysosomes contain abundant levels of the putative receptor, lgp96 (Cuervo and Dice, 1996
), or import
channels for substrate proteins but low levels of lysosomal
proteases, fusion into tubules might correct such imbalances.
; Koning et al., 1993
). Indeed, serum-supplemented nonconfluent cells are rapidly dividing, while serum-deprived cells are not.
).
In these studies, 83% of the ly-hsc73 was localized in the
lysosomal lumen and 17% on the lysosomal membrane.
).
). Whatever its mode of entry, Fig. 8 shows that
[3H]hsc73, if it is first purified by ATP-affinity chromatography, is very stable to intralysosomal hydrolysis. The biochemical features of hsc73 that render it resistant to lysosomal proteolysis are not known, but this protein stability
must contribute to its steady state localization within lysosomes.
showed that only 20% of endocytosed HRP was found in a postlysosomal supernatant after
cell fractionation on sucrose gradients. This result includes
the fraction of the endocytosed HRP released by ruptured
endosomes and lysosomes during cell fractionation. It
should be noted that even if 20% of the mAb 13D3 did escape from the vacuolar apparatus into the cytosol, it would
block only ~4% of the cytosolic hsc73, which is an abundant cytosolic protein (Giebel et al., 1988
; Terlecky and
Dice, 1993
).
) and the ER (Vogel et al., 1990
; Nicchitta and Blobel, 1993
; Panzner et al., 1995
). In addition, ly-hsc73 could aid in the digestion of proteins once they have entered lysosomes, since other molecular chaperones can
facilitate degradation of substrate proteins (Sherman and
Goldberg, 1992
). Degradation of endocytosed [3H]RNase
A was not affected by the presence of large amounts of endocytosed mAb 13D3 (Fig. 6). Nevertheless, it remains possible that the digestion of proteins is facilitated by hsc73
only if the proteins enter the lysosome from the cytosol.
Fig. 9.
Model representing the roles of hsc73 and ly-hsc73 in
the selective lysosomal protein degradation pathway. The protein
substrate depicted is RNase A, and the black rectangle represents
the KFERQ sequence. Steps 1-4 are as described in the text.
[View Larger Version of this Image (19K GIF file)]
). In addition, the fraction of hsc73 associated with the
lysosomal membrane (Fig. 5 D) might participate in this
step. For RNase A, there is a kinetic intermediate in the
import process in which 2 kD of the carboxyl terminus remains outside of the lysosome (Cuervo et al., 1994
).
; Vogel et al., 1990
; Nicchitta and Blobel, 1993
; Panzner et al., 1995
). In this case,
the action of the ly-hsc73 might require repeated rounds of
peptide binding and ATP hydrolysis. Whether or not sufficient intralysosomal ATP exists for such an action remains
to be established. Alternatively, intralysosomal hsc73 may
facilitate protein import in some fashion not dependent on
ATP hydrolysis. For example, hsc73 and substrate proteins
might enter lysosomes as a complex. Finally, the substrate
protein is released inside the lysosome and degraded. It remains possible that ly-hsc73 also facilitates this process,
but such a role could not be demonstrated using endocytosed [3H]RNase A.
). The
second model predicts that intraorganellar hsp70s act as
translocation motors (Glick, 1995
). In this case, the hsc70s
must be attached to the organellar membrane, and, through
conformational changes driven by cycles of ATP hydrolysis, the polypeptide is simultaneously unfolded and pulled
inside the organelle. Further studies are required to distinguish between these models and to elucidate additional
features of this selective lysosomal proteolytic pathway.
Received for publication 6 November 1995 and in revised form 12 March 1997.
1. Abbreviations used in this paper: hsc, heat shock cognate; hsp, heat shock protein; lgp, lysosomal glycoprotein; NCS, newborn calf serum; PBP, peptide binding protein; PTA, phosphotungstate; SSA1p, stress 70 subgroup A gene 1 product.We thank Dr. Joseph Chandler for his generous gifts of mAb 13D3; Dr. David Albertini, Dr. Joan King, Mr. Marc Shaffel, and Mr. Rob Wilson for technical advice regarding the confocal microscopy; and Dr. Laura Liscum and Ms. Marilyn Negron for critical review of the manuscript.
This work was supported by grant AG06116 from the National Institutes of Health.