* Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235; The Department
of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea; and the § Department of Medicine and
Physiology, University of Tennessee, Memphis, Tennessee 38163
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the number of pathologies known
to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition
and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular
structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin,
Hsp70 and Hsp90 (70- and 90-kD heat shock protein,
respectively) concentrate in HEK 293 and HeLa cells.
The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with -tubulin, an established centrosomal marker.
Density gradient fractions containing purified centrosomes are enriched in proteasomal components and
cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For
example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition
of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively
recruits the cytosolic pools of ubiquitin, Hsp70, PA700,
PA28, and the 20S proteasome. Thus, the centrosome
may act as a scaffold, which concentrates and recruits
the systems which act as censors and modulators of the
balance between folding, aggregation, and degradation.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MANY pathologies result from the inability of a mutant protein to properly fold (Thomas et al.,
1995). It is therefore important to understand
the mechanisms by which misfolded proteins are recognized, processed, and ultimately degraded by the cell. The
26S proteasome catalyzes the intracellular degradation of
damaged or misfolded proteins, the rapid degradation of
short-lived regulatory proteins such as those involved in
cell cycle control and signaling, as well as the generation of
antigenic peptides for presentation to MHC-I (Coux et al.,
1996
). Degradation of most substrates by the proteasome
requires the covalent conjugation of ubiquitin, an event
catalyzed by the sequential action of three enzymes
(Ciechanover, 1994
; Hochstrasser, 1996
). The ubiquitination machinery acts progressively, resulting in a polyubiquitin chain that serves as a signal for degradation of the
modified protein by the 26S proteasome.
The 26S proteasome is composed of two major subcomplexes. The 20S proteasome, a 700-kD cylinder, constitutes the catalytic core of the protease (Coux et al., 1996;
Baumeister et al., 1998
). Structural (Lowe et al., 1995
;
Groll et al., 1997
) and biochemical evidence (Wenzel and
Baumeister, 1995
) indicate the catalytic sites are located
within a hollow cavity of the cylinder. Access to these sites
is achieved through narrow openings at the ends of the cylinder which may be gated by regulatory proteins. This topology limits substrates to short peptides and completely
unfolded proteins.
The regulatory complex of the 26S proteasome is a 20-subunit, 700-kD activator called PA7001 or 19S cap (Coux
et al., 1996), which associates with either (or both)
end(s) of the 20S proteasome to form the 26S complex (Yoshimura et al., 1993
; Coux et al., 1996
; Adams et al.,
1997
). Such binding probably activates proteasome function by opening access to the central cavity, thereby increasing access of substrates to the catalytic sites. PA700
has multiple ATPase activities (Coux et al., 1996
), binds to
and cleaves polyubiquitin (Lam et al., 1997
), and functions
to enhance degradation of proteins, perhaps by unfolding
and/or translocating its substrates to the central cavity (Ma
et al., 1994
; Adams et al., 1997
).
A second regulator of the 20S proteasome is PA28 or
11S, a 180-kD heterohexameric ring-shaped complex that
binds to the proteasome in an orientation similar to that of
PA700 (Dubiel et al., 1992; Ma et al., 1993
; Gray et al.,
1994
). PA28 does not require ubiquitinated substrates for
activity and functions to increase proteasomal processing
of short peptides rather than larger proteins in vitro. Synthesis of PA28 is stimulated by
-interferon, implicating its probable functional importance in antigen production and
presentation (Realini et al., 1994
; Ahn et al., 1995
; Dick
et al., 1996
). New data suggest the proteasome may exist in
vitro in a complex simultaneously with PA28 and PA700,
although the physiological relevance of such an observation is unclear (Hendil et al., 1998
).
A role has been established for the proteasome in the
degradation of incompletely folded or misfolded proteins
(Kopito, 1997). For example, both cystic fibrosis (CF)-
causing mutant forms (Riordan et al., 1989
) and up to 70%
of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) (Ward and Kopito, 1994
; Jensen
et al., 1995
; Ward et al., 1995
) have been shown to be degraded in an ATP-dependent, ubiquitin/proteasome-mediated manner (Jensen et al., 1995
; Ward et al., 1995
). CFTR
is a member of the ATP-binding cassette (ABC) supergene family of membrane transport proteins (Higgins,
1992
), and consists of five domains, including two nucleotide binding domains (NBD1 and NBD2), two transmembrane domains (TMD1 and TMD2), and a PKA-sensitive regulatory domain (R) (Riordan et al., 1989
).
Notably, of the more than 800 CF-causing CFTR mutations identified to date, several initiate the CF pathology
by affecting the ability of the nascent polypeptide to fold
into a functional, stable native state (Qu et al., 1997). One
such mutation, the deletion of phenylalanine 508 (
F508)
is by far the most common, accounting for ~70% of the
disease causing alleles (Tsui, 1992
). This mutation, located
in NBD1, results in a folding defective protein which is incompletely glycosylated and fails to efficiently traffic to
the apical membrane (Cheng et al., 1990
), and is degraded by the proteasome (Jensen et al., 1995
; Ward et al., 1995
).
Several other CF-causing folding mutations (Gregory et al.,
1991
; Sheppard et al., 1996
) including P205S which is located in the third membrane spanning helix (Wigley et al.,
1998
) of TMD1, also result in inefficient processing and
maturation and increased degradation.
Significantly, both the F508 and P205S folding mutants
are functional when they assume a native conformation,
thus raising the possibility that overcoming the maturation
deficiency by correcting the underlying folding defect or
by circumventing a proteolytic recognition step may be of
therapeutic benefit. Unfortunately, inhibition of the proteasome in HEK 293 cells expressing either wild-type or
F508 CFTR results in the accumulation of polyubiquitinated CFTR including insoluble, perhaps aggregated, species (Jensen et al., 1995
; Ward et al., 1995
).
Heat shock proteins, such as Hsp70 and Hsp90, have
been shown to play a role in both preventing protein aggregation and promoting folding or degradation of a wide
range of proteins. Recently, the ubiquitination of certain
proteins has been shown to depend upon Hsp70, implicating it in the targeting of misfolded or mutant proteins for
degradation by the proteasome (Bercovich et al., 1997). In
this regard, Hsp70 has been shown to interact transiently with immature, incompletely folded CFTR (Yang et al.,
1993
). While this paper was under review, a report appeared that demonstrated that the Hsp90 inhibitor geldanamycin prevents the association of immature CFTR with
the chaperone, blocks its maturation, and enhances its
degradation by the proteasome (Loo et al., 1998
).
In spite of the large body of evidence demonstrating the proteasome machinery's involvement in the degradation of a diverse range of substrates, it is unclear whether its functions are distributed homogeneously throughout the cell or if they are segregated. We present evidence herein of the centrosome as a unique location in which the proteasome machinery, Hsp70, and Hsp90 are concentrated in resting HEK 293 and HeLa cell lines. In addition, the centrosome, a structure thought previously to function primarily during cell division, responds to inhibition of the proteasome and increases in the level of misfolded proteins by expanding and recruiting cellular pools of the proteasome components and Hsp70.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Plasmids pCMVNot6.2 and pCMVNot6.2-F containing expressible human CFTR cDNAs were the generous gift of Dr. Johanna Rommens (The
Hospital For Sick Children, Toronto). Two anti-human (COOH terminus) CFTR antibodies were used: mouse monoclonal antibody 24-1 from
Genzyme Diagnostics and rabbit polyclonal antibody R3194 (Zeng et al.,
1997
). Mouse monoclonal anti-rat Grp78 (BiP) was obtained from StressGen Biotechnologies Corp. Monoclonal anti-ubiquitin and anti-
-tubulin
antibodies and rhodamine-conjugated wheat germ agglutinin were purchased from Sigma Chemical Co. Mouse monoclonal anti-Hsp70 and anti-Hsp90 antibodies were from Affinity Bioreagents, Inc. Goat polyclonal
anti-aldolase was from Biodesign International. Mouse monoclonal anti-
lamin B1 antibody was from Zymed Laboratories, Inc. Mouse monoclonal
anti-
-cop antibodies contained in media from secreting hybridoma cells
were the generous gift of Dr. George Bloom (Department of Cell Biology,
University of Texas Southwestern Medical Center). Fluorescein-conjugated concanavalin A was from Molecular Probes, Inc. Fluorescently labeled secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Lactacystin was purchased from Calbiochem. All other
materials were of the highest quality commercially available.
Generation and Characterization of Antisera
Polyclonal anti-PA28 (Ma et al., 1993), anti-PA28-
(Song et al., 1996
),
and anti-20S proteasome (Ma et al., 1994
) were generated as described.
Polyclonal antibodies were prepared in chickens against highly purified
bovine PA700. Chicken IgY was purified from egg yolks of immunized
birds. The antibodies specifically recognized multiple subunits of the
PA700 complex, including p112, p97, p58, p56, and p45, upon Western
blot analysis of crude cell lysates and could specifically immunoprecipitate
PA700 from solution using an agarose bound anti-IgY antibody. Polyclonal antibodies were prepared in rabbits against an HPLC-purified peptide representing the 16 COOH-terminal amino acids of human p31
(Nin1p), a subunit of PA700 (Kominami et al., 1995
).
Preparation of P205S Mutant Expression Construct
Oligonucleotide-directed mutagenesis as described (Andrews and Lesley,
1998) was used to generate the mutant CFTR from the parent expression
vector pCMVNot6.2. In brief, mutants were selected based upon the incorporation of a second-site mutation in
-lactamase which alters its substrate specificity leading to resistance of transformed bacteria to cefotaxime and ceftriaxone in addition to ampicillin. The sequence of the
mutagenic primer used to create P205S was 5'-CGTGTGGATCGCTTCTTTGCAAGTGGC-3'. Incorporation of the mutation was verified by
DNA sequencing. Transfection-quality plasmid DNA was prepared using
reagents supplied by Qiagen Inc.
Cell Culture and Transfection
Wild-type and mutant CFTR cDNAs were transfected into human embryonic kidney (HEK) 293 or HeLa cells (American Type Culture Collection) using the Fugene Mammalian Transfection Reagent (Boehringer Mannheim). Cell lines were maintained in DME supplemented with 10% FCS, 50 µg/ml streptomycin, and 50 units/ml penicillin.
Preparation of Centrosomes
Centrosomes were isolated from HEK 293 and HeLa cells by discontinuous gradient ultracentrifugation according to the method of Moudjou and
Bornens (1998). In brief, cells in the exponential phase of growth were
treated with 1 µg/ml cytochalasin D and 0.2 µM nocodazole for 1 h. Cells
were collected by trypsinization and centrifugation and the resulting pellet
was washed in TBS followed by 0.1× TBS/8% sucrose. Cells were resuspended in 2 ml of 0.1× TBS/8% sucrose followed by addition of 8 ml lysis
buffer (1 mM Hepes, pH 7.2, 0.5% NP-40, 0.5 mM MgCl2, 0.1%
-mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 1 mM
PMSF). The suspension was gently shaken and passed five times through
a 10-ml narrow-mouth serological pipette to lyse the cells. The lysate was
spun at 2,500 g for 10 min to remove swollen nuclei, chromatin aggregates,
and unlysed cells. The resulting supernatant was filtered through a nylon
membrane followed by addition of Hepes buffer and DNase 1 to a final
concentration of 10 mM and 1 µg/ml, respectively, and incubated on ice
for 30 min. The mixture was gently underlaid with 1 ml of 60% sucrose solution (10 mM Pipes pH 7.2, 0.1% Triton X-100, and 0.1%
-mercaptoethanol containing 60% [wt/wt] sucrose) and spun at 10,000 g for 30 min to
sediment centrosomes onto the cushion. The upper 8 ml of the supernatant was removed and the remainder, including the cushion, containing the concentrated centrosomes was gently vortexed and loaded onto a discontinuous sucrose gradient consisting of 70, 50, and 40% solutions from
the bottom, respectively, and spun at 120,000 g for 1 h. Fractions were collected and stored at -70°C before further analysis.
SDS-PAGE and Western Blotting
Density gradient fractions were diluted into 1 ml of 10 mM Pipes, pH 7.2, and centrosomes were sedimented at 14,000 rpm in a microfuge for 15 min
at 4°C. Centrosome pellets were resuspended in Laemmli sample buffer
containing 5% -mercaptoethanol and electrophoresed on 10% SDS-PAGE gels. Proteins were transferred onto nitrocellulose membranes in
the presence of Towbin transfer buffer (25 mM Tris, 192 mM glycine, and
20% methanol, pH 8.3). Membranes were blocked in TBS-T (Tris-buffered saline/0.1% Tween-20) containing 10% nonfat dry milk for 1 h and
then incubated in fresh blocking buffer containing primary antibody at the
desired concentration. Membranes were washed several times in TBS-T
followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized
with enhanced chemiluminescence.
Inhibition of 20S Proteasome
The proteolytic activity of the endogenous proteasomal pool was inhibited by the addition of the potent fungal product lactacystin. The inhibitor was added to 293 cells 48-72 h after transfection at a concentration of 10 µM. Treatment of cells was carried out for 2 to 12 h and was immediately followed by several washes with PBS before fixation for immunocytochemistry.
Immunocytochemistry
For immunofluorescent subcellular localization of CFTR (Lee et al.,
1997), transiently transfected cells attached to glass coverslips were rinsed
three times with PBS followed by fixation and permeabilization with 1 ml
of ice-cold methanol for 10 min at -20°C. Upon removal of methanol, cells
were again rinsed three times with PBS and incubated 10 min in 1 ml of
PBS supplemented with 50 mM glycine (this and all subsequent manipulations were carried out at room temperature). This buffer was then removed and nonspecific sites were blocked with 0.1 ml of blocking medium (PBS supplemented with 5% goat serum, 1% BSA, and 0.1% gelatin) for
1 h in a humidified chamber. After blocking, the medium was replaced
with 0.1 ml of blocking medium containing a 1/100 dilution of the respective primary antibody for 1 h. Cells were next washed three times with
blocking medium and incubated for 1 h with blocking medium containing
a 1/100 dilution of the appropriate fluorescently labeled secondary antibody. For double-labeling experiments, these primary and secondary incubations were repeated with antibodies against the second protein of interest. For lectin affinity staining involving either fluorescently tagged
wheat germ agglutinin or concanavalin A, these reagents were added at
either a 1/250 dilution with the secondary antibody, or at 1 µg/ml in PBS
for 30 min before the initial fixation and permeabilization, respectively.
Fluorescent images were obtained using a Bio-Rad MRC 1024 confocal
microscope. Expression levels of cells expressing CFTR were divided into
two categories, those which required a gain of 1,150 or higher to visualize
(low expressers) and those which required a gain setting of 950 or less
(high expressers). In each case the Iris was held constant at 3.5. CMV-driven CFTR expression levels can be categorized as either low or high.
At the high expression level, some cells form CFTR inclusions without
lactacystin treatment. This is observed for the folding mutants as well as
for wild-type CFTR. This is likely due to an overload of the cellular folding machinery and may account, in part, for the low (~30%) maturation
efficiency observed for wild-type CFTR overexpressed in cultured cells
(Ward and Kopito, 1994
). We have therefore restricted our study to cells expressing low levels of CFTR.
Subcellular Morphometry
Due to periodic irregularities in the shape of the perinuclear structure, morphometric analysis describing their apparent diameter, as delineated by confocal imaging of immunostained HEK 293 cells, was limited to cells containing reasonably symmetric centrosomes. Measurements of diameter were performed using software supplied by the manufacturer. Three fields (×400) of each experiment were randomly selected and the diameters were measured from all the cells in each field. In the case of elliptically shaped structures, the geometric mean of the diameters were used. Data presented are reported as the mean ± SEM.
Analysis of Soluble and Insoluble Cellular Fractions
2.5 × 105 HeLa cells per 3-cm dish were either mock-transfected or transfected with P205S mutant CFTR expression plasmid. 48 h post-transfection, one of each was treated with 10 µM lactacystin for 12 h. Cells were washed with PBS, collected by trypsinization, and pelleted in a microfuge at 4°C. Each pellet was washed twice with PBS and resuspended in 100 µl PBS supplemented with Complete protease inhibitor cocktail (Boehringer Mannheim). After lysis by passage 10-20 times through a 27-gauge needle, lysates were spun at 14,000 g for 1 h at 4°C. The supernatants were collected and the pellets were washed in supplemented PBS followed by centrifugation at 14,000 g for 30 min. After aspiration of the wash solution, the pellets were resuspended in 100 µl supplemented PBS. Both pellets and supernatants were stored at -70°C until analysis. Equal volumes of each pellet and supernatant were analyzed by SDS PAGE through 4-20% gels followed by Western immunoblotting versus the indicated antibodies as described above.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Concentration of Proteasome Machinery at the Centrosome
A central question with respect to proteasome-mediated degradation of misfolded integral membrane proteins is where in the cell does proteolysis occur. To address this question, we employed immunocytochemistry to delineate the subcellular distribution of several key components of the proteasome pathway including: the 20S proteasome, the PA700 and PA28 activator complexes, ubiquitin, and Hsp70.
In HEK 293 cells, antibodies directed against each of these proteasome components identified multiple distinct subcellular pools. The 20S proteasome (Fig. 1, A and D), the PA700 complex (Fig. 1, B and E), and ubiquitin (Fig. 1 G) are each clearly discernible in both a nuclear pool and a cytoplasmic pool. In contrast, PA28-associated immunofluorescence was identified mainly in the nucleus (Fig. 1, C and F), and Hsp70 was primarily found in a reticular pattern in the cytosol (Fig. 1 H). In addition to these general distributions, staining with each of these specific antibodies revealed the existence of a unique perinuclear site in which all of the studied components concentrate (Fig. 1, A-H). The structure is surrounded by but does not colocalize with staining against the ER lumenal chaperone BiP (Fig. 1 F), and is proximal to the Golgi apparatus as illustrated by staining with the lectin, WGA (Fig. 1, D and E). These same subcellular distributions were also observed in HeLa, COS, and CHO cells suggesting generality of the observation (data not shown).
|
Further investigation of this perinuclear structure using
double-labeled immunofluorescence revealed exact colocalization of the 20S proteasome, PA700, PA28, ubiquitin,
and Hsp70 with -tubulin, an established centrosomal
marker (Mitchison and Kirschner, 1984
; Oakley and Oakley, 1989
). The majority of
-tubulin staining was restricted to the centrosome, although an expected faint diffuse cytosolic staining was also observed corresponding to
the soluble fraction of this protein. These data implicate
the centrosome as a unique site for the colocalization and
concentration of the proteasomal machinery and certain
cell stress chaperones under basal conditions, suggesting a
novel function for the centrosome.
The specificity of these proteasomal antibodies for immunocytochemistry is supported by several control experiments. First, using indirect immunofluorescence and confocal microscopy, staining with non- and preimmune
rabbit sera or secondary antibody alone revealed virtually
no detectable signal (data not shown). Second, preabsorption of chicken anti-PA700 antiserum against purified antigen completely eliminated immunocytochemical fluorescence (Fig. 1, I and J). Likewise, a similar preabsorption of
rabbit anti-PA28 and Western blot analysis demonstrated
the specificity of this antisera. And third, staining with
anti-p31, a rabbit polyclonal antibody directed against a
single subunit of PA700, yielded a staining pattern that is
indistinguishable from that generated with the chicken
anti-PA700 complex antibodies. Similarly, staining with
polyclonal antisera raised against a peptide from the subunit of PA28 (Song et al., 1996
) was identical to that obtained with whole anti-PA28 (data not shown).
To confirm our immunocytochemical observations, centrosomes were purified from nocodazole/cytochalasin
D-treated HEK 293 and HeLa cells by sucrose gradient
ultracentrifugation and subjected to Western blotting (Fig.
2). Analysis of the final purification step revealed a peak
of -tubulin immunoreactivity in fractions 3 through 6 corresponding to sucrose densities of between 50 and 60%. In
agreement with the immunocytochemical data, 20S proteasome, p31 (PA700), PA28, and Hsp70 were observed in
fractions containing
-tubulin, indicating their copurification with the centrosome in the absence of an intact cytoskeleton. In addition, Hsp90 also copurified with
-tubulin. Interestingly, Hsp70, Hsp90, and to a lesser extent
PA28, were also observed in lighter gradient fractions, where faint
-tubulin immunoreactivity was detected. This
observation may be explained either by a heterogeneous
population of centrosomes or lower-affinity binding of the
chaperones and PA28 to the centrosome relative to that of
20S and PA700. Sucrose fractions were devoid of other
subcellular markers, including BiP (ER), aldolase (cytosol), Lamin B1 (nucleus), and
-cop (Golgi), establishing
the purity of the centrosome preparations.
|
Expansion of the Centrosome in Response to Proteasome Inhibition
Treatment of cells with lactacystin, a potent and specific
inhibitor of the proteasome (Fenteany et al., 1995) resulted in a significant increase in size of the centrosome.
Representative images of cells treated and untreated with
lactacystin, were stained for PA28 and the lectin Con A
(Fig. 3, A and B). Similar results were obtained for cells
stained for 20S proteasome, Ub, PA700, Hsp70, and
-tubulin. These results were quantified by morphometric analysis of centrosome diameters (identified by fluorescent
staining for
-tubulin or perinuclear proteasomal components). Comparison of lactacystin-treated and untreated
HEK 293 cells revealed a twofold increase in mean diameter in response to the proteasome inhibition (Fig. 3 C).
These data suggest that the centrosome is a dynamic structure, capable of expansion in response to inhibition of the
proteasome. This expansion may be due to a build up of misfolded proteins, which would otherwise be degraded by
the proteasome concentrated at the centrosome. To further test this hypothesis, we expressed in HEK 293 cells
wild-type CFTR and two variants known to misfold,
namely
F508 and P205S, and examined their effect on
the centrosome and the proteasome machinery.
|
Expansion in Response to Expression of Mutant Protein
Consistent with previous reports (Cheng et al., 1990), immunolocalization of CFTR in transiently transfected HEK
293 cells clearly differentiates the wild-type CFTR subcellular locale from that of the folding mutants. Antiserum
directed against the COOH terminus of CFTR recognized
wild-type CFTR at the plasma membrane of transfected
cells (Fig. 4 A) and at an early stage of maturation that
colocalized with the ER-resident chaperone BiP (Fig. 4
B). The ER is the site of initial membrane translocation
and integration of nascent polytopic membrane proteins.
In sharp contrast,
F508 and P205S mutant CFTR are detected predominantly in the ER of transfected cells as illustrated by the ER pattern of CFTR staining (Fig. 4, C
and E) and the complete colocalization with BiP staining (Fig. 4, D and F).
|
Cells expressing low levels of the wild-type CFTR do
not significantly perturb either the PA28 distribution or
size of the centrosome, although in highly expressing cells
a fraction of CFTR colocalizes within this structure as seen
in Fig. 5 A. Similar results were obtained with other proteasome components studied (data not shown). In striking
contrast, expression of P205S (Fig. 5 B) or F508 (data not
shown) expands the centrosome in a manner similar to
that observed when cells are treated with lactacystin alone
(Fig. 3). This observation provides further support for the
accumulation of misfolded proteins at the site of the centrosome even when the proteasome is functional.
|
Recruitment of Proteasomal Components to a Centrosome-Associated Inclusion
In cells overexpressing mutant CFTR (P205S) and treated
with lactacystin, we observed the formation of large, perinuclear aggregates of misfolded CFTR which appear to
arise from the centrosome as indicated by colocalization
with -tubulin (Fig. 6). Identical results were observed
with
F508 expressing cells (data not shown). In addition,
a remarkable and comprehensive recruitment of the cytosolic pools of the 20S proteasome (Fig. 7 A), PA700 (B),
ubiquitin (D), and Hsp70 (E) to the site of CFTR aggregate formation was observed. This recruitment is coincident with a depletion of the cytosolic pools implicating this
as the source. PA28 is also recruited to the inclusion in response to the build up of misfolded CFTR (Fig. 7 C). Morphometric analysis of the CFTR aggregate formed in lactacystin treated cells is presented in Fig. 7 F. A composite of data from Fig. 3 E describing centrosome expansion due
to proteasome inhibition alone relative to nontreated cells
is included for comparison. The diameter of the aggregate
formed in lactacystin-treated cells expands to four to six
times the size of the centrosome from which it is apparently derived.
|
|
Next, to further investigate the observed redistribution
of the proteasomal machinery and Hsp70 in response to
the formation of lactacystin-induced intracellular inclusions of misfolded CFTR, we separated cell lysates into
soluble and insoluble cellular fractions. In mock-transfected cells, -tubulin was observed primarily in the soluble fraction (Fig. 8). However, in cells expressing P205S
mutant CFTR and treated with lactacystin,
-tubulin was observed distributed between the soluble and insoluble
fractions. PA28, PA700, and Hsp70 redistributed in a manner parallel to
-tubulin (Fig. 8). An intermediate degree
of redistribution was observed for mock-transfected cells
treated with lactacystin and untreated mutant transfected
cells (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The work reported here describes the immunocytochemical (Fig. 1) and biochemical (Fig. 2) identification of the centrosome as a unique subcellular location in which, under basal conditions, components of the proteasome proteolytic pathway and certain relevant heat shock chaperones concentrate. This localization was observed under normal growth conditions in HEK 293, HeLa, COS, and CHO cells. Moreover, the association of the proteasome with the centrosome does not require an intact F-actin nor microtubular network as indicated by their presence in the purified centrosomal fractions obtained from nocodazole/ cytochalasin D-treated cells (Fig. 2), indicating that their localization at this site is not simply a result of clustering at the minus-ends of microtubules. This does not, however, exclude the possibility that the cytoskeleton may be required for trafficking to and from this location.
When the cellular level of misfolded protein is high, either due to the overexpression of a misfolded mutant protein (such as F508 or P205S CFTR) or the inhibition of
the proteasome, the cell responds by expanding the diameter of the centrosome up to twofold (Figs. 3 and 5). Assuming a spherical, three-dimensional shape, this would
translate to more than a fourfold increase in its volume.
Under high loads of misfolded substrate and/or insufficient proteasome activity, the centrosome, Hsp70, and the
proteolytic machinery undergo correspondent redistribution to a sedimentable fraction (Fig. 8). The presence of
the perinuclear proteasome concentration in control cells
argues that the simplest explanation for this expansion involves the targeting of misfolded proteins to this centralized locale for rapid and efficient degradation. Consistent
with this hypothesis, the centrosomal-associated proteasomal machinery is active (Fabunmi, R.P., W.C. Wigley, P.J.
Thomas, and G.N. DeMartino, unpublished observations).
When degradation is insufficient the misfolded proteins
accumulate at and proximal to this site eventually forming
a large inclusion. In light of this finding and the proximity
of the centrosome to the Golgi and lysosomes, care should
be taken when interpreting subcellular localization of
overexpressed proteins.
It is particularly interesting that under the highest loads
of misfolded protein, when the proteasome is inhibited
and a mutant protein is overexpressed, the cell responds
by extensively recruiting additional proteasomal machinery from the cytosolic pools to the centrosome-associated
inclusion (Fig. 7). The function of the centrosome in nucleating and organizing microtubules (Mitchison and
Kirschner, 1984) suggests the involvement of microtubule-based motors in the recruitment process. Consistent with
this notion is the observation that the nuclear pools of proteasomal component remain unperturbed (Fig. 7). Further
experiments will be required to determine if the recruitment is an active process or simply due to diffusion although the latter possibility is unlikely because molecules
the size of the 26S proteasome are too large to simply diffuse through the cytosol (Janson et al., 1996
).
Similar questions exist as to the targeting of the substrates to this site. While this manuscript was under review, an interesting and complementary study reported
the formation of CFTR aggregates at the centrosomes
(Johnston et al., 1998). Formation of the aggregates required an intact microtubular system implicating a motor-based translocation of substrate to the centrosome,
analogous to the potential mechanism of proteasome recruitment suggested here. Interestingly, fibrillar extensions, as observed by CFTR and proteasomal staining
(Figs. 6 A and 7), radiate from the centrosomally localized aggregates. These fibrils are sensitive to treatment with
nocodazole suggesting their microtubular origin. However, the centrosomal localization of the aggregates and
associated proteasomal components persist in lactacystin
treated cells after nocodazole treatment. The mechanisms
employed for assembly and, potentially, disassembly of
these structures deserves further study. Regardless of the means of assembly, it is clear that this structure concentrates and recruits proteins that would be expected to perform
a censor function by monitoring and perhaps controlling
the balance between folding, degradation, and aggregation
of nascent membrane proteins in the cell.
Understanding the composition and assembly of the
proteasome-enriched centrosomes should provide new insight into the mechanisms of quality control employed by
eukaryotic cells. For example, it is unclear if PA700, free
or in complex with the 20S proteasome, participates directly in the recognition of the misfolded CFTR, altered
localization, or the formation of the inclusions. However, it is possible that free PA700 uses its poly-Ub binding domains and other cues to identify substrates not only for the
proteasome, but also for transport to the centrosome. Subsequently or coincidentally, ATP-dependent association
with the 20S proteasome, PA700-mediated Ub isopeptidase activity, and other likely activities such as active unfolding would then serve to reexamine the substrate and perhaps, as an alternative to degradation, allow a second
attempt at folding before degradation. Such iterative steps
are known to occur in the Hsp60 class of chaperones
(Bukau and Horwich, 1998) and the Clp family of proteases (Kessel et al., 1996
).
The accumulation of overexpressed mutant CFTR at
the centrosome upon inhibition of proteolysis suggests
that it may serve as a terminal point in the pathway of misfolded polypeptides suggesting that they either assemble
or nucleate at this location. It is interesting to note that
similar inclusions (Russell, 1890; Valetti et al., 1991
) have
been previously observed in heat-stressed (Vidair et
al., 1996
), protease-inhibited cells (Wójcik et al., 1996
; Johnston et al., 1998
), and a growing family of pathologies
related to protein misfolding such as Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and type-1 spinocerebellar ataxia (SCA1) (Bruijn et al., 1998
; Cummings et
al., 1998
; Sisodia, 1998
). The detailed structural and functional relationships among these inclusions are unknown
and warrants further investigation.
The centrosomal localization of the proteolytic machinery under basal conditions described in this study may play
an important role in the degradation of proteins involved
in progression through the cell cycle (King et al., 1996). In
addition, this position also places the proteasome and
chaperones proximal to the cellular organelles directly involved in the production, maturation, and trafficking of
membrane proteins, a strategic locale for machinery involved in the recognition and processing of mutant proteins. Interestingly, recent studies in yeast, which lack the
resolution of the current work, place the 26S proteasome
at either the nuclear envelope-endoplasmic reticulum
(Enenkel et al., 1998
) or, in fission yeast, at the nuclear periphery at rest and in a nuclear-associated spot during meiosis (Wilkinson et al., 1998
). Although the ER is generally
considered the quality control organelle, based on BiP,
WGA, and Con A staining, the current higher resolution
studies indicate that in mammalian cells the proteasome
concentrates in a compartment probably post-ER, lacking
the complex glycoproteins of the Golgi. Dissecting the biochemical function of the centrosome should reveal if it is
the site of proteasome-mediated degradation of misfolded
and mutant membrane proteins. We are actively addressing these issues.
![]() |
Footnotes |
---|
Address correspondence to Philip J. Thomas, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: (214) 648-8723. Fax: (214) 648-9268. E-mail: thomas07{at}utsw.swmed.edu
Received for publication 21 October 1998 and in revised form 25 March 1999.
We thank the members of the Thomas, DeMartino, and Muallem Laboratories for advice and helpful discussions; George Bloom and Elena Kaznacheyeva for excellent technical advice; and Helen Yin and Bruce Horazdovsky for helpful comments.
This work was supported by research grants from the American Heart Association (9740033N) and National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (NIDDK; DK49835) to P.J. Thomas, NIDDK (DK46181) to G.N. DeMartino, National Institute of Dental Research (NIDR; DE12309), and NIDDK (DK38938) to S. Muallem. P.J. Thomas is an Established Investigator of the American Heart Association.
![]() |
Abbreviations used in this paper |
---|
20S, 20S proteasome; cAMP, adenosine 3',5'-cyclic monophosphate; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; Hsp70/Hsp90, heat shock protein (70 and 90 kD, respectively); NBD, nucleotide binding domain; PA700/ PA28, proteasome activator complex (700 and 180 kD, respectively); PKA, cAMP-dependent protein kinase; SCA1, type-1 spinocerebellar ataxia; TMD, transmembrane domain; Ub, ubiquitin; TBS-T, tris-buffered saline/0.1% Tween-20.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Adams, G.M., S. Falke, A.L. Goldberg, C.A. Slaughter, G.N. DeMartino, and E.P. Gogol. 1997. Structural and functional effects of PA700 and modulator protein on proteasomes. J. Mol. Biol. 273: 646-657 |
2. |
Ahn, J.,
N. Tanahashi,
K. Akiyama,
H. Hisamatsu,
C. Noda,
K. Tanaka,
C.H. Chung,
N. Shibmara,
P.J. Willy,
J.D. Mott, et al
.
1995.
Primary structures of
two homologous subunits of PA28, a ![]() |
3. |
Andrews, C.A., and
S.A. Lesley.
1998.
Selection strategy for site-directed mutagenesis based on altered ![]() |
4. | Baumeister, W., J. Walz, F. Zuhl, and E. Seemuller. 1998. The proteasome: paradigm of a self-compartmentalizing protease. Cell. 92: 367-380 |
5. |
Bercovich, B.,
I. Stancovski,
A. Mayer,
N. Blumenfeld,
A. Laszlo,
A. Schwartz, and
A. Ciechanover.
1997.
Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70.
J. Biol.
Chem.
272:
9002-9010
|
6. |
Bruijn, S.,
M.K. Houseweart,
S. Kato,
K.L. Anderson,
S.D. Anderson,
E. Ohama,
A.G. Reaume,
R.W. Scott, and
D.W. Cleveland.
1998.
Aggregation
and motor neuron toxicity of an ALS-linked SOD1 mutant independent
from wild-type SOD1.
Science.
281:
1851-1853
|
7. | Bukau, B., and A.L. Horwich. 1998. The Hsp70 and Hsp60 chaperone machines. Cell. 92: 351-366 |
8. | Cheng, S.H., R.J. Gregory, J. Marshall, S. Paul, D.W. Souza, G.A. White, C.R. O'Riordan, and A.E. Smith. 1990. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell. 63: 827-834 |
9. | Ciechanover, A.. 1994. The ubiquitin-proteasome proteolytic pathway. Cell. 79: 13-21 |
10. | Coux, O., K. Tanaka, and A.L. Goldberg. 1996. Structure and functions of the 20S and 26S proteasomes. Annu. Rev. Biochem. 65: 801-847 |
11. | Cummings, C.J., M.A. Mancini, B. Antalffy, D.B. DeFranco, H.T. Orr, and H.Y. Zoghbi. 1998. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nature Genet. 19: 148-154 |
12. | Dick, T., T. Ruppert, M. Groettrup, P.-M. Kloetzel, L. Kuehn, U.H. Koszinowski, S. Stevanovic, H. Schild, and H.-G. Rammensee. 1996. Coordinated dual cleavage induced by the proteasome regulator PA28 lead to dominant MHC ligands. Cell. 86: 253-262 |
13. |
Dubiel, W.,
G. Pratt,
K. Ferrell, and
M. Rechsteiner.
1992.
Purification of an
11S regulator of the multicatalytic protease.
J. Biol. Chem.
267:
22369-22377
|
14. |
Enenkel, C.,
A. Lehmann, and
P.-M. Kloetzel.
1998.
Subcellular distribution of
proteasomes implicates a major location of protein degradation in the nuclear envelope-ER network in yeast.
EMBO (Eur. Mol. Biol. Organ.) J.
17:
6144-6154
|
15. | Fenteany, G., R.F. Standaert, W.S. Lane, S. Choi, E.J. Corey, and S.L. Schreiber. 1995. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science. 268: 726-731 |
16. | Gray, C.W., C.A. Slaughter, and G.N. DeMartino. 1994. PA28 activator protein forms regulatory caps on proteasome stacked rings. J. Mol. Biol. 236: 7-15 |
17. | Gregory, R.J., D.P. Rich, S.H. Cheng, D.W. Souza, S. Paul, P. Manavalan, M.P. Anderson, M.J. Welsh, and A.E. Smith. 1991. Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2. Mol. Cell. Biol. 11: 3886-3893 |
18. | Groll, M., L. Ditzel, J. Lowe, D. Stock, M. Bochtler, H.D. Bartunik, and R. Huber. 1997. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 386: 463-471 |
19. | Hendil, K.B., S. Khan, and K. Tanaka. 1998. Simultaneous binding of PA28 and PA700 activators to 20S proteasomes. Biochem. J. 332: 749-754 |
20. | Higgins, C.F.. 1992. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8: 67-113 . |
21. | Hochstrasser, M.. 1996. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30: 405-439 |
22. | Janson, L.W., K. Ragsdale, and K. Luby-Phelps. 1996. Mechanism and size cutoff for steric exclusion from actin-rich cytoplasmic domains. Biophysical J. 71: 1228-1234 [Abstract]. |
23. | Jensen, T.J., M.A. Loo, S. Pind, D.B. Williams, A.L. Goldberg, and J.R. Riordan. 1995. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell. 83: 129-135 |
24. |
Johnston, J.A.,
C.L. Ward, and
R.R. Kopito.
1998.
Aggresomes: a cellular response to misfolded proteins.
J. Cell Biol.
143:
1883-1898
|
25. | Kessel, M., W. Wu, S. Gottesman, E. Kocsis, A.C. Steven, and M.R. Maurizi. 1996. Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY. FEBS Lett. 398: 274-278 |
26. |
King, R.W.,
R.J. Deshaies,
J.-M. Peters, and
M.W. Kirschner.
1996.
How proteolysis drives the cell cycle.
Science.
274:
1652-1658
|
27. | Kominami, K.-I., G.N. DeMartino, C.R. Moomaw, C.A. Slaughter, N. Shimbara, M. Fujimuro, H. Yokosawa, H. Hisamatsu, N. Tanahashi, Y. Shimizu, et al . 1995. Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae. EMBO (Eur. Mol. Biol. Organ.) J. 14: 3105-3115 [Abstract]. |
28. | Kopito, R.R.. 1997. ER quality control: the cytoplasmic connection. Cell. 88: 427-430 |
29. | Lam, Y.A., W. Xu, G.N. DeMartino, and R.E. Cohen. 1997. Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature. 385: 737-740 |
30. |
Lee, M.G.,
X. Xu,
W. Zeng,
J. Diaz,
R.J.H. Wojcikiewicz,
T.H. Kuo,
F. Wuytack,
L. Racymaekers, and
S. Muallem.
1997.
Polarized expression of
Ca2+ channels in pancreatic and salivary gland cells.
J. Biol. Chem.
272:
15765-15770
|
31. |
Loo, M.A.,
T.J. Jensen,
L. Cui,
Y.-X. Hou,
X.-B. Chang, and
J.R. Riordan.
1998.
Perturbation of Hsp90 interaction with nascent CFTR prevents its
maturation and accelerates its degradation by the proteasome.
EMBO (Eur.
Mol. Biol. Organ.) J.
17:
6879-6887
|
32. | Lowe, J., D. Stock, B. Jap, P. Zwickl, W. Baumeister, and R. Huber. 1995. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268: 533-539 |
33. |
Ma, C.-P.,
J.H. Vu,
R.J. Proske,
C.A. Slaughter, and
G.N. DeMartino.
1994.
Identification, purification, and characterization of a high molecular weight,
ATP-dependent activator (PA700) of the 20 S proteasome.
J. Biol. Chem.
269:
3539-3547
|
34. |
Ma, C.-P.,
P.J. Willy,
C.A. Slaughter, and
G.N. DeMartino.
1993.
PA28, an activator of the 20S proteasome, is inactivated by proteolytic modification of its
carboxyl terminus.
J. Biol. Chem.
268:
22514-22519
|
35. | Mitchison, T., and M. Kirschner. 1984. Microtubule assembly nucleated by isolated centrosomes. Nature. 312: 232-237 |
36. | Moudjou, M., and M. Bornens. 1998. Method of centrosome isolation from cultured animal cells. In Cell Biology: A Laboratory Handbook. Academic Press, Inc., New York. 111-119. |
37. | Oakley, C.E., and B.R. Oakley. 1989. Identification of gamma tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. Nature. 338: 662-664 |
38. | Qu, B.H., E. Strickland, and P.J. Thomas. 1997. Cystic fibrosis: a disease of altered protein folding. J. Bioenerg. Biomem. 29: 483-490 . |
39. |
Realini, C.,
W. Dubiel,
G. Pratt,
K. Ferrell, and
M. Rechsteiner.
1994.
Molecular cloning and expression of a ![]() |
40. | Riordan, J.R., J.M. Rommens, B.-S. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J.-L. Chou, et al . 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 245: 1066-1073 |
41. | Russell, W.. 1890. Address on a characteristic organism of cancer. Brit. Med. J. 2: 1356-1360 . |
42. |
Sheppard, D.N.,
S.M. Travis,
H. Ishihara, and
M.J. Welch.
1996.
Contribution
of proline residues in the membrane-spanning domains of cystic fibrosis
transmembrane conductance regulator to chloride channel function.
J. Biol.
Chem.
271:
14995-15001
|
43. | Sisodia, S.S.. 1998. Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial? Cell. 95: 1-4 |
44. |
Song, X.,
J.D. Mott,
J. von Kampen,
B. Pramanik,
K. Tanaka,
C.A. Slaughter, and
G.N. DeMartino.
1996.
A model for the quaternary structure of the proteasome activator PA28.
J. Biol. Chem.
271:
26410-26417
|
45. | Thomas, P.J., B.-H. Qu, and P.L. Pedersen. 1995. Defective protein folding as a basis of human disease. Trends Biochem. Sci. 20: 456-459 |
46. | Tsui, I.-C.. 1992. The spectrum of cystic fibrosis mutations. Trends Genet. 8: 392-398 |
47. | Valetti, C., C.E. Grossi, C. Milstein, and R. Sitia. 1991. Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum. J. Cell Biol. 115: 983-994 [Abstract]. |
48. | Vidair, C.A., R.N. Huang, and S.J. Doxsey. 1996. Heat shock causes protein aggregation and reduced protein solubility at the centrosome and other cytoplasmic locations. Int. J. Hyperther. 12: 681-695 . |
49. |
Ward, C.L., and
R.R. Kopito.
1994.
Intracellular turnover of cystic fibrosis
transmembrane conductance regulator.
J. Biol. Chem.
269:
25710-25718
|
50. | Ward, C.L., S. Omura, and R.R. Kopito. 1995. Degragation of CFTR by the ubiquitin-proteasome pathway. Cell. 83: 121-127 |
51. | Wenzel, T., and W. Baumeister. 1995. Conformational constraints in protein degradation by the 20S proteasome. Nat. Struct. Biol. 2: 199-204 |
52. | Wigley, W.C., S. Vijayakumar, J.D. Jones, C. Slaughter, and P.J. Thomas. 1998. The transmembrane domain of CFTR: design, characterization and secondary structure of synthetic peptides m1-m6. Biochemistry. 37: 844-853 |
53. |
Wilkinson, C.R.M.,
M. Wallace,
M. Morphew,
P. Perry,
R. Allshire,
J.-P. Javerzat,
J.R. McIntosh, and
C. Gordon.
1998.
Localization of 26S proteasome
during mitosis and meiosis in fission yeast.
EMBO (Eur. Mol. Biol. Organ.)
J.
17:
6465-6476
|
54. | Wójcik, C., D. Schroeter, S. Wilk, J. Lamprecht, and N. Paweleta. 1996. Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome. Eur. J. Cell Biol. 71: 311-318 |
55. | Yang, Y., S. Janich, J.A. Cohn, and J.M. Wilson. 1993. The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc. Natl. Acad. Sci. USA. 90: 9480-9484 [Abstract]. |
56. | Yoshimura, T., K. Kameyama, T. Takagi, A. Ikai, F. Tokunaga, T. Koide, N. Tanahashi, T. Tamura, Z. Cejka, W. Baumeister, K. Tanaka, and A. Ichihara. 1993. Molecular characterization of the "26S" proteasome complex from rat liver. J. Struct. Biol. 111: 200-211 |
57. |
Zeng, W.,
M.G. Lee,
M. Yan,
J. Diaz,
I. Benjamin,
C.R. Marino,
R. Kopito,
S. Freedman,
C. Cotton,
S. Muallem, and
P. Thomas.
1997.
Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and
duct cells.
Am. J. Physiol.
273:
C442-C455
|