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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 146-155
Abnormal Expression and Subcellular Distribution of Subunit
Proteins of the AP-3 Adaptor Complex Lead to Platelet Storage Pool
Deficiency in the Pearl Mouse
By
Lijie Zhen,
Shelley Jiang,
Lijun Feng,
Nicholas A. Bright,
Andrew
A. Peden,
Albert B. Seymour,
Edward K. Novak,
Rosemary Elliott,
Michael B. Gorin,
Margaret S. Robinson, and
Richard T. Swank
From the Department of Molecular and Cell Biology, Roswell Park
Cancer Institute, Buffalo, NY; University of Cambridge, Cambridge
Institute for Medical Research, Cambridge, UK; Pfizer Central Research,
the Department of Genomics, Targets and Cancer, Groton, CT; and the
Departments of Human Genetics and Ophthalmology, University of
Pittsburgh, Pittsburgh, PA.
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ABSTRACT |
The pearl mouse is a model for Hermansky Pudlak Syndrome (HPS),
whose symptoms include hypopigmentation, lysosomal abnormalities, and
prolonged bleeding due to platelet storage pool deficiency (SPD). The
gene for pearl has recently been identified as the beta3A subunit of
the AP-3 adaptor complex. The objective of these experiments was to
determine if the expression and subcellular distribution of the AP-3
complex were altered in pearl platelets and other tissues. The beta3A
subunit was undetectable in all pearl cells and tissues. Also,
expression of other subunit proteins of the AP-3 complex was decreased.
The subcellular distribution of the remaining AP-3 subunits in
platelets, macrophages, and a melanocyte-derived cell line of pearl
mice was changed from the normal punctate, probably endosomal, pattern
to a diffuse cytoplasmic pattern. Ultrastructural abnormalities in
mutant lysosomes were likewise apparent in mutant kidney and a cultured
mutant cell line. Genetically distinct mouse HPS models had normal
expression of AP-3 subunits. These and related experiments strongly
suggest that the AP-3 complex regulates the biogenesis/function of
organelles of platelets and other cells and that abrogation of
expression of the AP-3 complex leads to platelet SPD.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE PEARL (pe) mutation arose
spontaneously in the C3H strain.1 The pearl (pe)
gene is inherited in an autosomal recessive manner and has been mapped
to chromosome 13.2-4 Mutant mice have oculocutaneous
pigment dilution with melanosomes both morphologically abnormal and
reduced in quantity.5 Platelet storage pool deficiency
(SPD) in pearl mutant platelets,6-8 like that of human
SPD,9 is characterized by deficiencies of the platelet
dense granule components serotonin and adenine nucleotides. In
addition, secretion of a third subcellular organelle, the lysosome, is
reduced in platelets and kidney.6,7 These phenotypes
indicate the pearl gene is involved in the biogenesis/function of all
three organelles. SPD in pearl mice leads to prolonged bleeding times, which can be corrected by marrow transplantation,10
indicating that the pearl gene acts in marrow progenitor cells. The
pearl mouse is one of a large group of mouse hypopigmentation mutants with accompanying inherited SPD.8 The molecular causes of
SPD are little understood.
Pearl is an established model for human Hermansky Pudlak syndrome
(HPS), an autosomal recessive inherited disease with the triad
phenotype of hypopigmentation, prolonged bleeding times due to platelet
SPD, and accumulation of ceroid pigment in lysosomal organelles.11-13 HPS is thus a disease of subcellular
organelles with misregulation of the biogenesis/function of the related
organelles, melanosomes, lysosomes, and platelet dense granules. The
syndrome occurs in diverse populations worldwide and causes
considerable morbidity and mortality due to a high incidence of
fibrotic restrictive lung disease, granulomatis colitis, and prolonged
bleeding.11,13
Additionally, pearl mice exhibit reduced sensitivity in the
dark-adapted state,14 have altered somatostatin binding to
the retina,15 and acceleration of retinal
apoptosis,16 suggesting a model for human congenital
stationary night blindness.
Intracellular protein sorting and trafficking are conducted by means of
carrier vesicles.17 The formation of a number of these
vesicles is mediated by heterotetrameric adaptor protein (AP)
complexes. Two types of AP complexes mediate the formation of
clathrin-coated vesicles.18 AP-1 recruits clathrin to
vesicles at the trans-Golgi network (TGN), whereas AP-2
performs a similar function at the plasma membrane. A third
adaptor-related coat complex, termed AP-3, probably facilitates
trafficking of vesicles from the TGN and/or endosomal compartments to
lysosomes and melanosomes.19-21 AP-3 is heterotetrameric
containing two large subunits, delta and beta3, a medium subunit, mu3,
and a small subunit, sigma3. In yeast, AP-3 is essential for
cargo-selective transport to the yeast vacuole
(lysosome).22-23 AP-3 is important for pigment granule biogenesis in Drosophila, as evidenced by the combined decrease in pigmentation and in expression of the AP-3 delta subunit in the
garnet mutant.19,24 Details of the function of AP-3
in mammals are less understood.
Recent positional/candidate cloning approaches have succeeded in the
molecular identification of the pearl gene. Mutant mice contain
nucleotide sequence changes in beta3A cDNA together with reductions of
beta3A transcript expression,25 indicating that the primary gene defect in pearl mice is in this subunit of the AP-3
complex. The present and related experiments show abnormal expression
and subcellular distribution of several subunits of this complex in
platelets and other tissues of pearl mice, and strongly suggest that
alterations of the AP-3 complex leads to SPD in these mice.
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MATERIALS AND METHODS |
Mice.
C57BL/6J pe/pe mutant mice, control C57BL/6J, C57BL/6J
pe/+, and C3H/HeJ mice together with other HPS mutant mice
[ruby eye (ru/ru), gunmetal (gm/gm), light ear
(le/le), muted (mu/mu), and sandy (sdy/sdy)]
were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were
subsequently bred and maintained in the animal facilities of Roswell
Park Cancer Institute.
Cell culture.
A melanocyte-derived cell line was obtained from epidermis of 1-day-old
pearl mice as described.26 Immortalized pearl cells have
been maintained in culture for more than 12 months. A control mouse
melanocyte line, melan-a, was kindly provided by Dr Dorothy Bennett (St
George's Hospital Medical School, London, UK).
Thioglycollate-stimulated macrophages were isolated and cultured as
described.27
Probes and antibodies.
The mu3A probe for Northern blotting was obtained from J. Pevsner (The Johns Hopkins University, Baltimore, MD).28
Polyclonal affinity-purified rabbit antibodies to the delta, beta3A,
beta3B, mu3A/B, and sigma3A subunits of the human AP-3 complex were
described by Simpson et al.19,29 The rabbit polyclonal
anti-rat lgp110 antiserum was previously described.30
Protein A conjugated to 15 nm colloidal gold was purchased from the
Department of Cell Biology, University of Utrecht (Utrecht, The Netherlands).
Immunoblots.
Fresh tissue or tissues snap frozen in liquid nitrogen were homogenized
in a proteinase inhibitor cocktail.31 The homogenized tissues were boiled in sample buffer and 40 µg protein was
electrophoresed on sodium dodecyl sulfate (SDS) polyacrylamide gels and
transferred to nitrocellulose in Western blotting. Blots were probed
with indicated primary antibodies prepared in rabbits, followed by peroxidase-labeled goat anti-rabbit IgG secondary antibody (Kirkegaard and Perry, Gaithersburg, MD), visualization with ECL reagent (Amersham, Piscataway, NJ), and exposure to x-ray film.
Immunofluorescence.
Cells were fixed in methanol followed by acetone at
 20°C, then labeled with an affinity-purified rabbit
antiserum raised against the delta subunit of the AP-3 complex
expressed as a fusion protein19 followed by
fluorescein-conjugated donkey anti-rabbit IgG.
Endocytosis of bovine serum albumin (BSA)-gold.
Ten nanometer colloidal gold was prepared by tannic acid/tri-sodium
citrate reduction of gold chloride.32 The colloid was adjusted to pH 5.5 with NaOH and conjugated to sufficient BSA to afford
protection from NaCl-induced flocculation. BSA-gold was harvested using
ultracentrifugation protocols which yielded monodisperse preparations
free of aggregates and unbound protein.33,34 The
preparations were dialyzed against phosphate-buffered saline (PBS) and
adjusted to an A520 of 1.4 with PBS. One milliliter of
BSA-gold was added to 4 mL Dulbecco's Modified Eagle Medium (DMEM) + 10% fetal calf serum, and cells grown to  80% confluence were
incubated with the conjugate-containing medium for 4 hours at 37°C
followed by incubation in conjugate-free medium for 20 hours as
previously described.35
Transmission electron microscopy (TEM).
For TEM, cells were removed from tissue-culture flasks by
trypsinization and pelleted in a bench-top microfuge at 500g
for 2 minutes. Cells or tissue sections were fixed with 2.5%
glutaraldehyde/2% paraformaldehyde in 0.1 mol/L Na cacodylate buffer,
pH 7.2, for 3 hours at room temperature and processed as described
previously.35 The sections were observed in a Philips CM
100 transmission electron microscope (Philips Electron Optics,
Cambridge, UK) at an operating voltage of 80 kV.
Immunoelectron microscopy (immuno-EM).
Cells were prepared for immuno-EM as described by
Griffiths.36 Cells were washed with PBS and fixed with 4%
paraformaldehyde/0.1% glutaraldehyde in 250 mmol/L HEPES, pH 7.2, at
room temperature for 1 hour. The cells were processed for immuno-EM as
described previously35 and ultrathin frozen sections were
collected from the knife edge using a mixture of sucrose and
methylcellulose. Immunolabeling of lgp110 was performed using the
protein A-gold technique37 as described
previously.35
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RESULTS |
The beta3A protein is greatly reduced in tissues of pearl mice.
Our recent studies25 showed that beta3A transcripts of
pearl mice are significantly decreased in quantity and are predicted to
produce a beta3A protein with a truncation of 130 amino acids of the
C-terminus of the 1,105-amino acid subunit. These results suggested
that expression and function of the beta3A protein would be
significantly affected in pearl tissues. This was confirmed by
immunoblotting of five tissues of pearl and control C57BL/6J mice
(Fig 1) using a polyclonal antibody against
human beta3A.19 The 130-kD beta3A protein
subunit is expressed in all control tissues examined, although
quantitative variation was observed. Concentrations were relatively
high in normal macrophages and platelets and intermediate in bone
marrow and liver and low in heart. Very low levels were detected in
normal kidney (not shown). In contrast, the beta3A subunit was not
detected in any tissue of pearl mice. Further, forms of beta3A of
altered size/mobility were not apparent.
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| Fig 1.
The beta3A protein is expressed at very low/undetectable
levels in platelets, macrophages and other tissues of pearl mice.
Extracts (40 µg protein) from indicated tissues of mutant pearl mice
and control C57BL/6J mice were analyzed by Western blotting. Sizes of
MW standard proteins are indicated at right. Blots were reprobed with
antibody to mouse actin (below) as a loading control. The high MW
material observed near the gel origin in this experiment in macrophage
extracts of normal and pearl mice was not reproducible.
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Levels of other subunits of the AP-3 heterotetramer are affected in
certain tissues of pearl mice.
The loss of one subunit of a protein complex can lead to decreased
stability of other subunit proteins. Expression of the other three
subunits of AP-3 was accordingly assessed in pearl by immunoblotting
(Fig 2). Levels of the 160-kD
delta subunit were consistently reduced to about half normal levels in
platelets from the pearl mutant (Fig 2A). Levels of the delta subunit
were also significantly diminished in bone marrow and eye of pearl mice
(not shown). The mu3A subunit was more drastically affected. Very small
amounts of mu3A subunits (molecular weight [MW], 45 kD) and
significantly reduced levels of sigma3A subunits (MW, 26 kD) were
visible in pearl platelets. Similar results were observed in mutant
bone marrow, eye, liver, and macrophages (mu3A) and bone marrow and eye
(sigma3A) (not shown). In control experiments, no alterations in
platelet levels of the gamma and sigma 1 subunits of another adaptor
complex, AP-1, were observed in pearl mice (not shown).
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| Fig 2.
Concentrations of other subunits of the AP-3 complex in
platelets (A) and brain (B) from the pearl mutant mouse. Samples (40 µg protein) were blotted and treated with antibodies to beta3A,
beta3B, delta, mu3A, sigma3A, and control actin.
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Although loss of one subunit of a protein complex can destabilize other
proteins of the complex, no effects on transcript levels of other
subunit components are expected. In fact, no alterations in levels of
the mu3A transcript were observed in several pearl tissues (kidney,
heart, lung, bone marrow, eye, and macrophages) by Northern blotting
(not shown). Thus, as expected, the observed reductions in levels of
the AP-3 subunits in pearl tissues are apparently restricted to subunit
proteins rather than transcripts.
Two forms of the beta3 subunit of the AP-3 complex have been described.
One is the beta3A subunit,19,20 and the second is betaNAP
or beta3B.38 In humans the two subunits are 61% identical at the amino acid level.19 Beta3A is ubiquitously expressed while expression of beta3B is restricted to neural tissue. Brain is
unique among all tissues in that both beta3A and beta3B subunits are
expressed therein. Therefore, it was predicted that, unlike other
tissues, the remaining subunits of the AP-3 tetramer would occur at
normal or near-normal levels in pearl brain since these subunits would
form active heterotetramers with the beta3B subunit, despite the
absence of beta3A. Immunoblotting (Fig 2B) confirmed this prediction.
Beta3A subunit levels were nondetectable; however, there were normal
levels of the beta3B, mu3A, delta, and sigma3A subunits in pearl brain.
Subcellular distribution of the AP-3 complex is altered in pearl
cells.
The subcellular distribution of the AP-3 complex was measured by
indirect immunofluorescence, using a polyclonal antibody19 to the AP-3 delta subunit (Fig 3). An
intense punctate distribution was apparent in normal C57BL/6J
platelets. In contrast, a weaker and more disseminated signal was
apparent in platelets of pearl mice. Likewise, a punctate distribution
of AP-3 in peripheral and perinuclear regions of macrophages and
melan-a cells, a mouse melanocyte line, was observed, similar to its
localization in other types of cells.19,20 The peripheral
pattern is consistent with an endosomal distribution. However, in pearl
primary macrophages and in a melanocyte-derived cell line from pearl
mice, this punctate distribution was abolished and only a weak, diffuse
cytoplasmic staining appeared. Thus, even though the delta subunit is
expressed in pearl cells, it appears to be unable to be recruited onto
membranes as seen in normal cells. Therefore, the AP-3 complex in these cells is both missing the beta3A subunit and is mislocalized, likely
resulting in a nonfunctional complex.
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| Fig 3.
The AP-3 delta subunit has a weak, diffuse localization
pattern in pearl cells. Immunofluorescence was conducted on melan-a
cells, a normal melanocyte cell line (A), on pearl melanocyte-derived
cells (B), on macrophages of normal (C) and pearl (D) mice, and on
platelets of normal (E) and pearl (F) mice. Scale bar = 10 µm.
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Genetic studies in yeast have shown that alkaline phosphatase, a
membrane protein normally residing in the vacuole, is missorted in
AP-3-deficient cells.22,23 To determine whether lysosomal membrane proteins are missorted in pearl cells, the cells were allowed
to endocytose BSA coupled to 10 nm gold for 4 hours, which was chased
for 20 hours to concentrate the probe in lysosomes. Frozen thin
sections of such cells were then labeled with an antibody against the
lysosomal membrane protein lgp11030 indirectly coupled to
15 nm gold. Figure 4a shows that the two
labels are associated with the same organelles, indicating that the
steady-state distribution of lgp110 in pearl cells is normal, although
it is possible that it arrives at its destination via a different
trafficking pathway.
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| Fig 4.
Endocytosed BSA-gold accumulates in lgp110-positive
electron-dense lysosomes. (a through c) Pearl cells were allowed to
endocytose BSA-10 nm gold for 4 hours followed by a chase in
conjugate-free medium for 20 hours. Gold accumulated in electron dense
lysosomes which could be immunolabeled with antibodies to the lysosomal
marker lgp110 (15 nm gold). Although most of the lysosomes were of
normal appearance (a), some were filled with rolled-up membranous
inclusions (b and c; arrowheads indicate the 15 nm gold-labeled
lgp110). (d through f) Plastic sections of BSA-10 nm gold-containing
lysosomes, showing the internal membranes. Scale bar = 200 nm.
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Intriguingly, some of the lysosomes in the pearl cells were found to
have an unusual appearance, filled with what appear to be rolled-up
membranous inclusions forming hollow cylinders (Fig 4b through f). We
have observed similar structures in primary cultures of fibroblasts
from mocha mice in which AP-3 function is likewise lost,39
although at a lower frequency (data not shown). Thus, they may reflect
abnormalities in lysosome function resulting from the AP-3 deficiency.
Morphologically abnormal subcellular granules were likewise observed in
kidney proximal tubule cells of pearl mice
(Fig 5). Although similar structures were
occasionally observed in kidney proximal cells of normal mice (Fig 5,
inset) they were smaller and less numerous than those of pearl kidney. Depressed rates of secretion of kidney lysosomal enzymes have been
observed in the pearl mutant.7,8 Likewise, kidney lysosomes with related morphological abnormalities similar to those of pearl (Fig
5) have been observed in other mouse HPS mutants related to
pearl.8,40 Thus, the abnormal granules in pearl kidney likely represent engorged lysosomes that accumulate in pearl kidney to
a greater extent than in normal kidney as a result of depressed secretory rates.
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| Fig 5.
Abnormal lysosomal morphology in kidney proximal tubules
of the pearl mutant. Kidneys from normal (A) and pearl (B) mice were
examined by standard TEM. Inset shows a rare small multilamellar
lysosome in normal tissue. Scale bar = 500 nm.
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Morphologically abnormal dense granules, typical of those observed in
SPD, have been described in platelets of pearl mice. Whole-mount
electron microscopy of air-dried unfixed platelets6 combined with studies of mepacrine-labeled platelet dense
granules,41 indicated that dense granules of pearl
platelets are present but are essentially "empty" with very low
contents of dense granule components such as serotonin and adenosine
diphosphate (ADP). However, no additional subcellular or other
morphological abnormalities of pearl platelets were apparent upon
standard transmission electron microscopic analyses (not shown).
Other mouse SPD/HPS models have normal concentrations of beta3A.
A significant number of mouse hypopigmentation mutants have organellar
phenotypes similar to that of the pearl mutant and are likewise models
for platelet SPD.8 Platelets, which are probably the most
intensively characterized cell in all these mutants, typically have
abnormalities in contents and/or secretion of dense granules and
lysosomes. Acccordingly, platelet extracts of five of these mutants
[ruby eye (ru/ru), gunmetal (gm/gm), light ear
(le/le), muted (mu/mu), and sandy (sdy/sdy)]
were analyzed by immunoblotting, using an antibody specific for the
beta3A subunit (Fig 6). It is apparent that
all of these mutants have levels of beta3A approximately equivalent to
that of their respective control strains. In other experiments (not
shown), concentrations of the delta, mu3A, and sigma3A AP-3 subunits
were likewise determined to be normal in platelet extracts of the same
mutants. Therefore, despite phenotypic similarities among these mouse
HPS-like mutants, altered expression of proteins of the AP-3 complex
appears to be specific to pearl. This result corroborates a large body
of evidence that these mutants are genetically unique.8 In
contrast, another mouse HPS-like mutant, mocha, has recently been found to contain mutations in the delta subunit of the AP-3 complex together
with decreased expression of other AP-3 complex proteins.39
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| Fig 6.
The beta3A subunit is present at normal levels in other
mouse HPS mutants. Platelet extracts (40 µg protein) were Western
blotted and probed with antibody to beta3A. The C57BL/6J strain is the
control strain for congenic mutants pearl (pe/pe), ruby eye
(ru/ru), gunmetal (gm/gm), and light ear
(le/le). The muted (mu/mu) and sandy (sdy/sdy)
mutants are maintained on other inbred backgrounds and therefore the
appropriate genetic control strains are mu/+ and
sdy/+, respectively. Blots were reprobed with an antibody to
mouse actin (below) as a loading control.
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DISCUSSION |
These experiments indicate that alterations in the AP-3 complex cause
platelet SPD. The primary mutation in pearl, a mouse model for platelet
SPD,6,8 occurs in the gene for the beta3A subunit of the
AP-3 complex.25 The present studies show that expression of
the beta3A subunit protein is completely or nearly completely
eliminated in several tissues, including platelets, of pearl mice. Loss
of beta3A occurs at both the transcript25 and protein
levels. Further, by immunofluorescent microscopy a remaining subunit,
delta, of the AP-3 complex is mislocalized subcellularly in several
pearl tissues including platelets. Such a substantial loss of beta3A,
combined with altered AP-3 subcellular distribution, would be expected
to abrogate normal AP-3 function in pearl mice and lead to platelet SPD.
The absence of beta3A in all tissues of pearl mice analyzed by Western
blotting suggests that the loss is systemic. This is consistent with
other evidence for multitissue involvement in both mouse8
and human HPS/SPD.12
The likelihood that AP-3 function is lost in pearl tissues is supported
by immunoblotting evidence for a nearly complete loss of the mu3A
subunit. The beta and mu subunits of the AP-1 and AP-2 complexes have
been shown to interact using the yeast two-hybrid system,42
and the loss of the mu3 subunit in the pearl tissues indicates that
without such an interaction, the mu subunit is unstable and gets
degraded. The gamma and sigma1 subunits of the AP-1 complex and the
alpha and sigma2 subunits of the AP-2 complex were also found to
interact in the two-hybrid system,42 and the delta and
sigma3 subunits of the AP-3 complex appear to interact as well because
they can be coimmunoprecipitated from pearl cell cytosol (A.A.P. and
M.S.R., unpublished observations). However, immunofluorescence microscopy (Fig 3) shows that the delta subunit in
pearl is mislocalized and fails to associate with membranes, indicating
that the partial complexes formed from the delta and sigma subunits are
not functional. An exceptional tissue is brain where normal levels of
other AP-3 subunits occur in pearl mice. The probable explanation is
that the brain specific beta3B subunit effectively substitutes for the
loss of the beta3A subunit (Fig 2), thus stabilizing the complex in
mutant mice.
The phenotypes of the pearl mutant suggest novel roles for the AP-3
complex in SPD and in regulation of granule structure, biogenesis, and
function of organelles in mammalian tissues. Pearl mice have
abnormalities in structure and/or function of at least three related
organelles: lysosomes, melanosomes, and platelet dense granules.
Lysosomes from several tissues of pearl mice are significantly altered
in function. Constitutive secretion of lysosomes from kidney proximal
tubule cells of pearl mice is reduced to one third the normal
rate7 and thrombin-mediated secretion of lysosomal enzymes
from platelets occurs at half the normal rate.6 The
abnormal lysosomal structures detected in melanocyte-derived cells and
kidney of pearl mice in these experiments suggest a morphological basis
for the lysosomal secretion defect. Structurally related organelles
containing unusual membranous multilamellar-like material have been
observed in melanocytes derived from patients with HPS.43
Also, related abnormal structures have been detected in kidney of
several other mouse HPS/SPD mutants,40 suggesting these
structures may be a general feature of genes causing HPS/SPD. Further,
these structures may be relevant to the kidney failure noted in a
subgroup of HPS patients.11 Increased rates of synthesis of
lysosomal enzymes have been observed in kidney of the pearl mutant.7 Retinal pigment epithelium of pearl mice contains fewer melanosomes that are morphologically larger and contain irregular
boundaries with clumps of melanin.5 Pearl melanosomes are
mislocalized, being absent within the apical processes of retinal
pigment epithelial cells. Also, the basal membrane of this tissue lacks
infoldings.5 The dense granules of pearl platelets are
grossly abnormal in content6 (see below). Together, these
phenotypes indicate that the AP-3 complex regulates all three
organelles. Such a role for AP-3 is consistent with converging genetic
and cell biological evidence of similarities in biosynthesis, composition, and regulation of these organelles.8,44
The importance of the AP-3 complex in the biosynthesis of melanosomes
is supported by the fact that mutations in the delta subunit of
Drosophila AP-3 produce the garnet eye pigment
phenotype.19-24 Garnet flies, like pearl
mice,5 have reduced eye pigmentation leading to aberrant
eye pigment color.
The detailed mechanism(s) by which reductions in AP-3 cause the granule
abnormalities characteristic of the pearl phenotype are undefined.
However, recent studies in both yeast and mammals suggest possible
scenarios. Selective deletions of yeast AP-3 subunits abolish transport
of alkaline phosphatase and the vacuolar t-SNARE, Vam3p, to the yeast
lysosome or vacuole.22,23 In mammals, the AP-3 complex
similarly regulates targetting to the lysosomal membrane of the
lysosomal integral membrane proteins LAMPI and LIMPII45 and
is necessary for in vitro formation of synaptic vesicles from
endosomes.46 Alterations in these vesicle budding and cargo
selection functions of AP-3 may cause the abnormalities of lysosomes,
melanosomes, and platelet dense granules of pearl mice. Further, the
AP-3 complex is located in the trans-Golgi network and in more
peripheral endosomal regions of the cell,19,20 sites within
the generally agreed subcellular pathway for biogenesis/trafficking of
lysosomal proteins. These locations are also consistent with observed
defects in constitutive and stimulated secretion of lysosomal contents
from kidney7 and platelets6 of pearl mice,
because lysosomal enzyme secretion may involve retrograde transport
from lysosomes through the prelysosomal/late endosomal
compartment.35,47 Detailed molecular studies indicate that
the AP-3 complex recognizes both tyrosine20,48 and
dileucine22,49,50 signals on lysosomal proteins during
vesicle budding and cargo selection.
Normal platelet activation releases at the site of a wound massive
quantities of serotonin and ADP, stored in dense granules, a process
critical for normal clotting. The near absence of these components in
dense granules leads to platelet SPD and prolonged bleeding
times.8,9 Therefore, it is important to understand the
relationship of AP-3 abnormalities and the diagnostic "empty" platelet dense granules of SPD observed in pearl mice.6 It may be reasonably speculated that this relationship is similar to that
observed between AP-3 deficiencies and lysosomal mistargetting. That
is, critical receptors/transporters of serotonin and adenine nucleotides may not be properly captured by a defective AP-3 complex during biosynthesis of the platelet dense granule membrane. This mutant
platelet phenotype may mimic the observed mistargetting of Vamp-3 and
alkaline phosphatase to the vacuole in yeast AP-3 mutants.22,23 It is likewise consistent with the
finding45 that proper targetting of the lysosomal membrane
proteins to lysosomes requires the AP-3 complex. Receptor-deficient
dense granules would ultimately lead to the "empty" dense
granules of SPD. It is now possible to directly test whether
mistargetting of important granule membrane proteins occurs in pearl
platelets and other cells.
The importance of the AP-3 complex in SPD and in the
biosynthesis/function of lysosomes, melanosomes, and platelet dense
granules is further documented by results of recent studies on the
mouse mocha hypopigmentation mutant. Mocha mice, arising from a
mutation on mouse chromosome 10, have abnormalities in and greatly
reduced expression of the delta subunit of the AP-3 complex and
accompanying reduced expression of other AP-3 subunits.39
Like pearl mice, mocha mice present with platelet SPD, and the effects
of the mocha mutant on intracellular organelles are similar to those of
pearl.8 In addition, mocha mice display an interesting
hyperactive phenotype.39,51 It is likely that the
hyperactive phenotype does not occur in pearl because the presence of
the beta3B subunit retains normal levels of the AP-3 tetramer (Fig 2)
in pearl brain. In contrast, no alternative delta subunit is expressed
in brain. Therefore, AP-3 subunits and function are absent in mocha
brain.39 The availability of both pearl and mocha mutants,
defective in expression of the beta3A or delta subunits, respectively,
of AP-3 should allow definitive tests of the role of AP-3 in SPD,
subcellular trafficking, and related processes in mammals.
Recently the gene responsible for the major form of human HPS common in
Puerto Rico has been cloned.52,53 This gene is ubiquitously
expressed and encodes a novel protein predicted to be
membrane-associated and 700 amino acids in size. The pale-ear mouse is
the appropriate mouse homologue for this form of human HPS.53,54 The pale-ear, pearl, and mocha HPS/SPD genes have related phenotypic effects on subcellular organelles8 as
does the recently cloned beige gene,55 and their protein
products may interact with each other or with the same targetting
signals. Therefore, it is possible that all four genes may regulate the same or closely related pathways of organelle biogenesis/function.
The phenotypic heterogeneity of HPS in humans suggested that the
disease is caused by multiple primary gene defects. Genetic heterogeneity is supported by the fact that a large number (14) of
separate mouse hypopigmentation genes cause HPS-like
phenotypes8 and, more directly, by reports of HPS patients
who have no discernible mutations in the recently identified human HPS
gene.56,57 In fact, two brothers with HPS and with
molecular alterations in the beta3A subunit have recently been
identified.58 Therefore, the pearl mouse is an appropriate
animal model for these patients. Additional and corresponding mouse HPS
mutants will be required to model other HPS patients accurately.
HPS is often a very debilitating disease due to SPD with associated
prolonged bleeding, colitis, and fibrotic lung disease, which leads to
increased mortality13 in the third to fifth decades of
life. Because no curative therapies exist, improved diagnostic and
therapeutic approaches would be welcome. The beta3A gene is now an
obvious target for these studies.
| |
ACKNOWLEDGMENT |
Dr Dorothy Bennett kindly provided the melan-a cell line. We thank
Joanne Pazik for aid in culturing of melanocyte lines and Michael E. Rusiniak and Edward P. O'Brien for helpful suggestions. We are
grateful to Dr Lawrence Pinto (Northwestern University, Evanston, IL)
and Dr Stephen Hardies (University of Texas, San Antonio) for their
help in earlier characterizations of the pearl mutant.
| |
FOOTNOTES |
Submitted December 29, 1998; accepted February 22, 1999.
L.Z., S.J., and L.F. contributed equally to this study.
Supported by National Institutes of Health Grants No. HL31698, HL51480,
and EY12104 (R.T.S.), by grants from the Medical Research Council and
the Wellcome Trust (M.S.R.), Grants No. EY09192 and EY0898 and grants
from The Eye and Ear Foundation of Pittsburgh (M.B.G.), Grant No.
HD28623 (R.E.), and by shared resources of the Roswell Park Cancer
Center Support Grant (P30 CA16056).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Richard T. Swank, PhD, Roswell Park Cancer
Institute, Elm and Carlton St, Buffalo, NY; e-mail:
rswank{at}mcbio.med.buffalo.edu.
| |
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ABSTRACT
INTRODUCTION