Regulation of tyrosinase trafficking and processing
by presenilins: Partial loss of function by familial
Alzheimer’s disease mutation
Runsheng Wang*, Phuong Tang*†, Pei Wang*‡§, Raymond E. Boissy¶, and Hui Zheng*†‡?
*Huffington Center on Aging and Departments of†Molecular and Human Genetics and‡Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030; and¶Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, OH 45267
Communicated by Xiaodong Wang, University of Texas Southwestern Medical Center, Dallas, TX, November 10, 2005 (received for review October 25, 2005)
Presenilins (PS) are required for ?-secretase cleavage of multiple
type I membrane proteins including the amyloid precursor protein
and Notch and also have been implicated in regulating intracellular
protein trafficking and turnover. Using genetic and pharmacolog-
ical approaches, we reveal here a unique function of PS in the
pigmentation of retinal pigment epithelium and epidermal mela-
nocytes. PS deficiency leads to aberrant accumulation of tyrosinase
(Tyr)-containing 50-nm post-Golgi vesicles that are normally des-
tined to melanosomes. This trafficking is ?-secretase-dependent,
and abnormal localization of Tyr in the absence of PS is accompa-
nied by the simultaneous accumulation of its C-terminal fragment.
Furthermore, we show that the PS1M146V familial Alzheimer’s
disease mutation exhibits a partial loss-of-function in pigment
synthesis. Our results identify Tyr and related proteins as physio-
logical substrates of PS and link ?-secretase activity with intracel-
lular protein transport.
?-secretase ? pigmentation ? melanocyte ? knock-out ? knock-in
A?40 and A?42, peptides that constitute the principal components
of the ?-amyloid plaques characteristic of Alzheimer’s disease (1).
Mutations in PS1 and PS2 lead to autosomal dominant inheritance
of familial Alzheimer’s disease (FAD). More than 100 mutations
have been identified in PS1 and PS2, and they spread throughout
the entire molecules. These mutations are known to alter the
regulated intramembrane proteolysis (4), PS are required for
processing and signaling of Notch (5), and this pathway likely
contributes to various PS developmental activities (6).
The PS-dependent ?-secretase activity requires the formation of
a high-molecular-weight complex containing nicastrin, Aph1,
and Pen2 (6). The active complex is assembled in a sequential and
interdependent manner through the endoplasmic reticulum and
Golgi compartments and requires posttranslational modifications.
The ?-secretase cleavage is preceded by extracellular processing
and exhibits relaxed sequence specificity. Nicastrin recently was
shown to function as the receptor for the ?-secretase substrates (7).
Besides APP and Notch, PS has been implicated in the processing
of a growing list of type I membrane proteins (reviewed in ref. 8).
However, with the exception of Notch, the physiological signifi-
cance of these proteolytic events remains speculative.
In addition to its ?-secretase activity, PS has been documented
to regulate intracellular protein trafficking (9). PS1 deficiency has
been reported to result in aberrant trafficking and maturation of
APP (10, 11), Notch (12, 13), tyrosine kinase receptor TrkB (14),
et al. (20) and Wilson et al. (21) implicated a role of PS1 in the
turnover of telencephalin and ?- and ?-synucleins, respectively.
They showed that these molecules accumulate in degradative
ammalian presenilins (PS) consists of two homologous pro-
by ?-secretase inhibitor treatment (20, 21). Thus, this activity is
likely mediated through a ?-secretase independent mechanism.
Similar to APP and Notch, tyrosinase (Tyr) (monophenyl mo-
nooxidase, EC 18.104.22.168), along with two Tyr-related proteins,
Tyr-related protein-1 (Tyrp1) and 3,4-dihydroxyphenylalanine
(DOPA) chrome tautomerase?Tyr-related protein-2 (DCT?
Tyrp2), are type I membrane proteins specialized in pigment
synthesis (22). Tyr catalyzes the conversion of tyrosine to DOPA,
which is an essential and rate-limiting step in melanin synthesis.
and DCT?Tyrp2 result in the synthesis of eumelanin (23). These
reactions are conducted within the melanosomes of vertebrate
and epidermal melanocytes. Melanosomes are endosomal?
lysosomal-related organelles. They progress through a series of
coated endosomes lacking pigmentation, to stages II, III, and IV
melanosomes, which are striated with increased melanin contents
(24). Under normal conditions, Tyr is glycosylated through the
secretory pathway, budded off from the trans-Golgi network as
50-nm vesicles, and transported to melanosomes to undergo pig-
ment synthesis and further maturation (25, 26).
pigment synthesis. Mutations in tyrosinase (Tyr) and Tyrp1 result in
retention of immature Tyr in the endoplasmic reticulum and are
associated with oculocutaneous albinism type 1 and 3, respectively
(27). In addition to the melanocyte-specific proteins, numerous
other molecules, including both highly conserved adaptor proteins,
soluble N-ethylmaleimide sensitive factor attachment protein re-
ceptors, and small GTPases of the Ras superfamily, as well as
unique vertebrate-specific protein complexes termed biogenesis of
lysosome-related organelles complexes, are known to regulate Tyr
trafficking and pigment processes (reviewed in refs. 24 and 28). In
addition to melanosomes, these proteins also play important roles
in regulating other endosomal?lysosomal-related organelles that
are essential for immune or neuronal functions.
In this work, we reveal an indispensable role of PS in Tyr
trafficking and pigmentation by a ?-secretase-dependent mecha-
FAD mutation in vivo.
Materials and Methods
Abs and Inhibitors. Rabbit polyclonal antisera against Tyrp1
(?PEP1), DCT?Tyrp2 (?PEP8), and C- and N-terminal Tyr
Conflict of interest statement: No conflicts declared.
Abbreviations: PS, presenilin; APP, amyloid precursor protein; FAD, familial Alzheimer’s
disease; RPE, retinal pigment epithelium; DOPA, 3,4-dihydroxyphenylalanine; Tyr, tyrosi-
AP3, adaptor protein-3; HPS, Hermansky–Pudlak syndrome; CTF, C-terminal fragment;
DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester.
§Present address: Department of Developmental Biology, Stanford University, Stanford,
?To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
January 10, 2006 ?
vol. 103 ?
no. 2 ?
been implicated to subject to similar PS-dependent intracellular
domain production and cellular signaling (reviewed in ref. 45).
Alternately, ?-secretase cleavage has been proposed to degrade
membrane proteins and possibly attenuate signaling pathways (8,
46). The well-established enzymatic activities of Tyr and related
proteins make them unlikely cell signaling molecules. Because the
C-terminal sequences of these proteins contain melanosomal tar-
geting signals (41–43), breakage of the CTFs by ?-secretase is
expected to disrupt their melanosomal localization and melanin
here to degrade these membrane proteins. As such, the accumu-
lation of CTFs of Tyr family of proteins in the absence of PS would
represent degradation intermediates, which otherwise may escape
detection due to PS-mediated proteolysis. Future studies are re-
quired to identify the cellular sites of CTF accumulation and to
Support for a function of PS in intracellular protein degrada-
tion also came from recent studies by Wilson et al. (21) and by
Esselens et al. (20), which documented delayed turnover and
subsequent accumulation of ?- and ?-synucleins and telencepha-
lin, respectively, in degradative organelles in PS1-null cells.
However, because these effects are not seen by ?-secretase
inhibitor treatment and because full-length proteins, not the
CTFs, accumulate, these are likely mediated through indepen-
In summary, we identify here a unique function of PS in melanin
synthesis and pigmentation. We present data that it is mediated by
its intracellular transport of post-Golgi Tyr-containing vesicles and
that this effect is ?-secretase dependent. We document a partial
loss-of-function activity by the PS1 M146V FAD mutation. These
findings raise the intriguing possibility that a compromised post-
Golgi vesicle transport may contribute to Alzheimer’s disease
We thank G. Martin and B. Sopher (University of Washington, Seattle)
for PS1M146V knock-in mice, R. Swank (Roswell Park Cancer Institute,
Buffalo, NY) for HPS mouse tissues, and A. Houghton (Memorial
Sloan–Kettering Cancer Center, New York) for the Tyr expression
vector. We thank V. J. Hearing (National Institutes of Health, Bethesda)
for the generous gifts of anti-Tyr, Tyrp1, and DCT?Tyrp2 and Pmel17
Abs; and H. Xu and P. Greenguard (The Rockefeller University, New
York) for the AB14 Ab. We thank Claire Haueter for expert EM
assistance. This work was supported by National Institutes of Health
Grants NS40039 (to H.Z.), AG20670 (to H.Z.), and AR45429 (to
R.E.B.) and by the Ellison Medical Foundation (H.Z.). R.W. and P.T.
are trainees of National Institutes of Health Training Grant T32
AG000183. H.Z. is a Zenith Award recipient from the Alzheimer’s
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