From the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6082
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ABSTRACT |
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Distinct cytoplasmic sorting signals target
integral membrane proteins to late endosomal compartments, but it is
not known whether different signals direct targeting by different
pathways. The availability of multiple pathways may permit some cell
types to divert proteins to specialized compartments, such as the
melanosome of pigmented cells. To address this issue, we characterized
sorting determinants of tyrosinase, a tissue-specific resident protein of the melanosome. The cytoplasmic domain of tyrosinase was both necessary and sufficient for internalization and steady state localization to late endosomes and lysosomes in HeLa cells. Mutagenesis of two leucine residues within a conventional di-leucine motif ablated
late endosomal localization. However, the properties of this
di-leucine-based signal were distinguished from that of CD3 The maintenance of morphological and functional integrity of
individual compartments of the secretory and endosomal pathways requires proper sorting of integral membrane components. Eukaryotic cells have evolved mechanisms to divert specific integral membrane protein components from several sites into tubular and/or vesicular carriers destined for late endosomes and lysosomes (reviewed in Refs. 1
and 2). Most of these mechanisms require the recognition of specific
sorting signals within the cytoplasmic domains of resident proteins by
cytosolic factors. It is becoming increasingly clear, however, that
there are redundant pathways to late endosomes and lysosomes in many
eukaryotic cells. An example of this redundancy is observed in
Saccharomyces cerevisiae, in which two apparently independent pathways with distinct effectors converge on the
lysosome-like vacuole to deliver different sets of hydrolases and
structural components (3, 4). Cargo selection for these two pathways relies on the recognition of distinct cytoplasmic sorting signals (5-8). Evidence also exists in mammalian cells for multiple pathways from the trans-Golgi network to late endosomes (9-13), but
sorting signals required for entry into different pathways have not yet been clarified.
What advantage would be imparted to an organism by the evolution of
independent pathways to late endosomes and lysosomes? For higher
eukaryotes, one potential advantage would be the ability to subvert one
pathway or the other in specific cell types to permit the development
of unique, tissue-specific lysosome-like organelles. Such a strategy
would be similar to that used to establish polarity in epithelial
cells. Protein sorting to the basolateral and apical cell surfaces
requires sorting signals that are recognized for packaging into
distinct vesicular carriers in both polarized and non-polarized cells
(14-16), but only in polarized cells are the vesicles targeted to
different destinations (reviewed in Ref. 17). Whether comparable
mechanisms act in protein sorting to other tissue-specific post-Golgi
organelles is not known.
One tissue-specific organelle with the potential for accumulating a
subpopulation of proteins diverted from the late endosome is the
melanosome, the organelle of melanocytes in which melanin is
synthesized (18, 19). Melanosomes resemble secretory lysosomes in that
they are highly acidic and often co-fractionate with markers of late
endosomes and lysosomes such as lamp1, lamp2, CD63, and acid
phosphatase (20-23). Furthermore, genes that control coat color in
mice and skin color in humans encode proteins that are involved in
lysosomal biogenesis (24, 25). Nevertheless, melanosomes contain a
unique cohort of resident membrane proteins and can be distinguished
from late endosomes and lysosomes in some pigmented cells (26). Thus,
resident proteins of both organelles must be differentially sorted, but
the characteristics that distinguish between sorting to late
endosomes/lysosomes and to melanosomes have not been established.
Perhaps the best characterized melanosomal resident protein in normal
and malignant melanocytes is tyrosinase (reviewed in Ref. 28). Human
tyrosinase, the rate-limiting enzyme in melanin biosynthesis, is a
melanocyte-specific, type I integral membrane glycoprotein with a large
lumenal domain, a 26-amino acid transmembrane domain, and a 30-amino
acid cytoplasmic domain (Ref. 27; see Fig. 1 for schematic). Impairment
of tyrosinase activity in humans results in oculocutaneous albinism
(OCA1; Ref. 29). Among the
many tyrosinase gene mutations that cause OCA or other pigmentation
defects, two affect the cytoplasmic domain. The tyrosinase gene in mice
homozygous for the cP (platinum)
allele contains a single base substitution that eliminates all but the
basic transmembrane anchor residues of the cytoplasmic tail; the
product of this allele is therefore mistargeted and expressed at the
cell surface (30). A similar mutation in human tyrosinase results in
OCA (31), but the localization of the gene product in melanocytes from
these patients has not been characterized. These reports suggest that
the cytoplasmic domain of tyrosinase is critical for melanosomal
protein sorting. Nevertheless, neither the precise nature of the
sorting signals nor their behavior in non-pigmented cells is
understood. Here, we explore the possibility that sorting signals with
unique characteristics direct tyrosinase into a sorting pathway that is
distinct from that used by typical residents of late endosomes and lysosomes.
The cytoplasmic domain of human tyrosinase contains several potential
conventional sorting signals. Among these are sequences that conform to
classic di-leucine-based (LL) motifs
(E510KQPLL515) and tyrosine-based motifs of the
YXXØ consensus sequence (in which a tyrosine, Y, is
separated by two residues, X, from an amino acid containing
a bulky hydrophobic side chain, Ø;
Y521HSL524), both common among proteins
localized to endosomal compartments (32-36). The cytoplasmic domain
also contains a second tyrosine-containing sequence
(Y525QSHL529) (see Fig.
1a). All three are conserved
in mouse tyrosinase, and the LL motif and Tyr525 are
conserved throughout vertebrate evolution. A role for the LL motif in
melanosomal sorting is supported by a requirement of LL residues in
tyrosinase related protein-1 (TRP-1) for intracellular localization
(37) and by recent evidence that the LL motif from mouse tyrosinase can
bind to the AP3 sorting complex in vitro (38). We postulated
that some or all of these motifs may direct endosomal sorting in a
non-pigmented cell type. Here, using a panel of chimeric proteins and
tyrosinase constructs in which these putative sorting signals have been
altered by mutagenesis, we characterize the trafficking of tyrosinase
in non-pigmented cells. Our results show that the LL motif is a
dominant signal that directs late endosomal sorting through a distinct
pathway from that directed by another well characterized late
endosomal LL-based signal. This suggests that non-pigmented cells
contain a second independent sorting pathway to the late endosome. Such a pathway provides a potential mechanism by which melanocytic cells can
differentially sort proteins to the melanosome.
Antibodies--
Wild-type and mutant forms of human tyrosinase
were recognized by Cell Culture and Transfections--
HeLa, CV-1, 293, and M1
cells (American Type Culture Collection) were maintained in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% heat-inactivated fetal bovine serum (Atlanta Biologics, Norcross,
GA) and antibiotics (100 units/ml penicillin, 100 µg/ml
streptomycin). Transient transfections of cells grown on coverslips in
6 well dishes were performed essentially as described (46) using
calcium phosphate precipitation. Except for overexpression studies,
50-100 ng of "expressor" plasmid DNA was used, and the balance was
made up with "carrier" plasmid (empty pCDM8.1 vector) to 5 µg
total; pilot studies showed that this protocol allowed detectable
expression of previously characterized lysosomally targeted proteins
without significant overexpression, as assessed by surface expression.
Cells were generally assayed 2-3 days following transfection. For
overexpression and competition experiments, cells were plated in 60- or
100-mm dishes and transfected with 8 or 20 µg of DNA, respectively.
Plasmids--
The R402Q allele of human tyrosinase was supplied
in the mammalian expression vector pCI (Promega, Madison, WI) by W. Storkus (University of Pittsburgh, Pittsburgh). Wild-type human
tyrosinase 501 (gift of S. Topalian, National Cancer Institute,
Bethesda), originally inserted into pCDNA3, was subcloned into
pCDM8.1 using EcoRI and XbaI restriction sites.
Original site-directed mutants were constructed using the tyrosinase
R402Q construct, but due to apparent misfolding in cells grown at
37 °C,2 the R402Q
substitution was corrected and the corrected form was used in all
experiments shown. Correction was performed by PCR-mediated amplification of the site-directed mutants of tyrosinase R402Q sequences downstream of the allelic variation (coding region for the
remaining lumenal domain fragment and the transmembrane and cytoplasmic
domains) and substitution of the amplicon into wild-type tyrosinase 501 cDNA using BglII and XbaI restriction enzyme
sites. The nucleic acid sequence of all constructs was confirmed by
dideoxynucleotide sequencing. The plasmids pCDM8.1 (47), human
invariant chain p33 (Ip33; Ref. 48), Tac (49) and the chimeric
constructs TTMb (46), TTL1 (50), and Tac-DKQTLL (originally referred to
as TT Site-directed Mutagenesis--
Most site-directed mutants
of the cytoplasmic tail of tyrosinase (see Fig. 1a) were
constructed using the GeneEditor in vitro Mutagenesis System
(Promega, Madison, WI) according to the directions of the supplier
(details available upon request). Constructs in which Tyr525
(XX/X/A) was mutated were made by PCR-mediated amplification of the wild-type or previously mutated sorting signals followed by
sub-cloning of the amplicon using a unique BglII recognition site in the tyrosinase coding region and an XbaI site in the
vector. Construction of Tac Chimeras--
All Tac chimeric constructs
were prepared as described (46) in the mammalian expression vector
pCDM8.1. Tac chimeras were generated for all constructs listed in Fig.
1 using a two-step PCR method (52). Briefly, a chimeric insert was made
encoding the lumenal and transmembrane domains of Tac fused to the
cytoplasmic domain of tyrosinase flanked by unique BglII and
XbaI restriction sites. The chimeric insert was then ligated
into a previously modified Tac chimeric construct. DNA sequences of all
chimeric constructs were confirmed by dideoxynucleotide sequencing.
Immunofluorescence Microscopy--
Forty-eight hours following
transfection, cells were fixed using 2% formaldehyde in PBS for 20 min
at room temperature and washed with PBS. In some cases, cells were
treated with cycloheximide (CHX; 50 µg/ml) for 1 h in
Dulbecco's modified Eagle's medium and washed in PBS prior to
fixation. Cells that had been transfected with DNA encoding Tac
chimeric proteins were either untreated or treated with 1 mg/ml
leupeptin for 4 h prior to fixation. Cells were stained as
described (53). Stained coverslips were mounted on microscope slides
using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL)
and visualized by fluorescence microscopy using a Zeiss Axiovert
microscope. Photographic images were digitized from negatives using a
Nikon LS-1000 slide scanner and Adobe Photoshop 3.0 software. For
overexpression studies and measurement of late endosomal diameter,
analyses were done using a Leica microscope fitted with a MicroMax
digital camera (Princeton Instruments Inc., Trenton, NJ) and OpenLab
software (Improvision, Coventry, UK). Statistical analyses of endosomal
diameter measurements was performed using the Mann-Whitney Rank Sum
Test on SigmaStat software (Jandel Scientific, San Rafael, CA).
Metabolic Labeling and Immunoprecipitation--
HeLa cells
transiently transfected with Tac chimeras or tyrosinase and/or Ip33
were metabolically labeled using 1-2 mCi/ml EXPRES35S (NEN
Life Science Products) for 30 min as described (50). Cell lysates
prepared with 1% Triton X-100 were subjected to immunoprecipitation using protein A-Sepharose bound to 7G7.B6 (anti-Tac), PIN.1
(anti-Ip33), or Antibody Internalization Assay--
HeLa cells transiently
transfected with Tac chimeras on coverslips were incubated with
purified 7G7.B6 anti-Tac antibody (50 µg/ml) in Dulbecco's modified
Eagle's medium, 10% fetal bovine serum for 30 min at 37 °C. All
coverslips were washed 3 times in PBS and fixed for 20 min in 2%
formaldehyde in PBS. Internalized mAb 7G7.B6 was detected using
LRSC-conjugated goat anti-mouse Ig, and total cellular Tac chimeras
were visualized by staining with rabbit anti-Tac and FITC-conjugated
goat anti-rabbit Ig as described above.
Sorting Signal Competition Assay--
This was performed
essentially as described (50). Tac chimeric proteins were purposefully
overexpressed in HeLa cells by transient transfection using 20 µg of
plasmid DNA in a 10-cm culture dish. In some experiments, cells were
co-transfected with lower levels (0.5 µg) of plasmid DNA encoding
Ip33 as a reporter protein. Sixty hours post-transfection, cells were
harvested and divided into aliquots for analyses. In some experiments,
levels of expression from each of the transfected DNAs was determined
by pulse metabolic labeling of 1 aliquot followed by
immunoprecipitation. For flow cytometric analysis, 0.5-1 × 106 viable cells were stained with the indicated primary
antibodies for 1 h, washed, and then stained with FITC- and
PE-conjugated secondary antibodies for an additional hour. Cells were
then washed, fixed in 2% formaldehyde in PBS, and analyzed using a
Becton Dickinson FACScan (Franklin Lakes, NJ) and CellQuest 1.2 software. Dead cells were excluded from analysis by forward and side
scatter measurements. In the analyses shown, cells were gated for those expressing high levels of the "overexpressor" Tac molecule at the
cell surface. Transfection efficiencies were monitored by parallel
immunofluorescence microscopy analyses.
Tyrosinase Localizes to Late Endosomes and Lysosomes in HeLa
Cells--
In order to assess the potential function of conventional
sorting signals in the cytoplasmic domain, it was first necessary to
determine the ultimate destination of tyrosinase in non-pigmented cells. To this end, tyrosinase was expressed in HeLa cells by transient
transfection, and its localization was assessed by indirect immunofluorescence microscopy (IFM).
When expressed at moderate levels, tyrosinase was detected in the
nuclear envelope and in reticular and vesicular structures both
throughout the cell cytoplasm and in the perinuclear area in most
transfected cells (Fig. 2a).
Reticular and nuclear envelope staining was variable and more intense
at higher levels of expression (data not shown). This staining pattern
would be consistent with localization to both the endoplasmic reticulum
(ER) and endosomal compartments at steady state. Prominent ER staining
is likely a result of slow egress of tyrosinase from the ER due to
inefficient folding, supported by pulse/chase analyses in which the
half-time for acquisition of resistance to digestion with
endoglycosidase H is longer than 1 h.2 To visualize
more clearly post-ER compartments, we therefore reduced the pool of
newly synthesized, ER-retained tyrosinase by treating transfected cells
with the protein synthesis inhibitor, cycloheximide (CHX).
As shown in Fig. 2b, the staining pattern of cells treated
for 1 h with CHX was predominantly vesicular. The appearance of tyrosinase in vesicular structures did not require the addition of
lysosomal protease inhibitors, suggesting that it is relatively stable
to proteolysis within these compartments. To determine the identity of
the structures, CHX-treated transfected cells were double-stained with
antibodies to tyrosinase and to endogenous endosomal proteins. As shown
in Fig. 3, a and b,
tyrosinase colocalized with lamp1, a constituent of late endosomes and
lysosomes. The staining pattern with both markers was nearly identical
in most cells at various expression levels. Near perfect colocalization was also observed with another marker of late endosomes and lysosomes, lamp2, but not with transferrin receptor (early and recycling endosomes), and peripheral tyrosinase+ vesicular structures did not
contain the cation-independent mannose 6-phosphate receptor (trans-Golgi network/late endosomes; data not shown). The
staining pattern for lamp1 in cells that expressed the transgene did
not consistently differ from that in cells that were not
transfected, indicating that tyrosinase expression at moderate levels
does not alter the morphology of lamp1-positive compartments. We
conclude that tyrosinase is directed to conventional late endosomes and lysosomes in HeLa cells. Similar observations were made in CV-1, M1,
and 293 cell lines (data not shown).
Role of Di-leucine and Tyrosine-based Sorting Signals in
Localization of Tyrosinase to Late Endosomes and Lysosomes--
To
characterize the cytoplasmic sequences responsible for targeting
tyrosinase to endosomal compartments, we used site-directed mutagenesis
to disrupt critical residues of the putative LL and YXXØ
sorting motifs and the additional conserved tyrosine residue Tyr525. Critical residues were altered individually or in
combination to alanine (Fig. 1a). Each mutagenized construct
was expressed in HeLa cells by transient transfection, and localization
was assessed in cells treated with CHX for 1 h by IFM and double
staining with antibodies to the tyrosinase transgene and to endogenous lamp1.
Tyrosinase with either or both of the cytoplasmic tyrosine residues
disrupted (LL/A/Y, LL/Y/A, and LL/A/A) demonstrated IFM staining
patterns similar to that of intact tyrosinase (Fig. 3, e, g,
and i). Reticular staining was variable, likely due to
differential retention in the ER, and relative staining intensities of
given vesicles for lamp1 and tyrosinase were often variable, but the patterns of lamp1- and tyrosinase-containing vesicles were identical for all three mutant forms (arrowheads, Fig. 3,
f, h, and j). In contrast, disruption
of the putative LL motif alone (AA/Y/Y) or deletion of the cytoplasmic
domain ( The Tyrosinase Cytoplasmic Domain Is Sufficient to Effect Late
Endosomal and Lysosomal Protein Sorting--
In order to rule out a
requirement for additional topological determinants that contribute to
sorting of tyrosinase, we transferred the cytoplasmic domain alone to a
reporter protein, Tac. Tac (the human interleukin-2 receptor
The results show that TTY behaved similarly to tyrosinase and was
localized to vesicular structures that overlapped nearly completely
with structures containing lamp1 (Fig. 4,
a and b). Furthermore, cells co-transfected with
both TTY and tyrosinase and treated with leupeptin demonstrated nearly
complete overlap of the two transgenes (data not shown). The staining
pattern of TTY was largely dependent on treatment of cells with
leupeptin (e.g. see Fig. 5),
consistent with degradation of the Tac lumenal domain in proteolytic
late endosomal compartments as implicated by parallel pulse/chase
experiments (data not shown). The localization of Tac chimeric proteins
with altered cytoplasmic tail residues paralleled that of the
corresponding tyrosinase mutant; disruption of the LL motif resulted in
cell-surface expression (Fig. 4, c and d),
whereas disruption of either (not shown) or both tyrosine residues had
no or subtle effects (Fig. 4, e and f). Residual vesicles associated with the altered LL motif mutants (TTY-AA/Y/Y and
TTY-AA/Y/A) often colocalized with lamp1 (Fig. 4, c and
d, and data not shown). Although this result could indicate
the existence of a secondary late endosomal sorting signal, the absence
of such colocalization with the corresponding full-length tyrosinase
constructs would support the alternative conclusion that these vesicles
represent the products of normal cell-surface protein turnover made
more apparent by leupeptin treatment. Unmodified Tac was expressed only
at the cell surface under these conditions (see Fig. 5), and chimeric
proteins containing the lumenal or transmembrane domains of tyrosinase
without the transmembrane domain were not localized to endosomal
compartments (data not shown), suggesting that only the cytoplasmic
domain contains independent endosomal sorting information. These data
demonstrate that the cytoplasmic domain of tyrosinase is both necessary
and sufficient for localization to conventional late endosomes and
lysosomes in HeLa cells, and these data confirm the role of the LL
motif in efficient endosomal protein sorting.
The Di-leucine Motif of Tyrosinase Functions as an Internalization
Signal at the Plasma Membrane--
Most post-Golgi sorting signals
also serve as internalization signals that function in efficient
retrieval of intracellular proteins from the cell surface. To determine
whether the tyrosinase cytoplasmic sorting signals function similarly,
we used IFM to assess the ability of the TTY chimeric proteins to
effect efficient internalization of anti-Tac antibody in live cells.
Chimeric proteins were transiently expressed in HeLa cells at moderate
levels, and anti-Tac mAb 7G7.B6 was added to the medium for 30 min at
37 °C to allow antibody uptake prior to fixation. Internalized
antibody was detected by staining with anti-mouse immunoglobulin, and
total cellular Tac chimeric protein was visualized using a rabbit
antiserum. As shown in Fig. 5 (a and b),
expression of TTY resulted in the efficient internalization of mAb
7G7.B6 and accumulation in intracellular vesicles. Although the assay
was not quantitative, the degree of internalization appeared similar to
that of TTL1 which contains the lamp1 cytoplasmic domain (Fig. 5,
i and j). Untransfected cells, assessed by lack
of staining with rabbit anti-Tac antibody, showed no detectable
internalization of the mAb. Furthermore, disruption of either or both
of the tyrosine residues in the cytoplasmic domain had no apparent
effect on internalization relative to the wild type (Fig. 5,
e and f and data not shown). In contrast,
disruption of the LL residues dramatically reduced internalization, as
evidenced by the intense cell-surface staining from 7G7.B6 in cells
transfected with either TTY-AA/Y/Y or TTY-AA/Y/A (Fig. 5, c
and d and data not shown). Interestingly, there was a small
degree of vesicular 7G7.B6 staining with both of these chimeras that
was not visible in cells transfected with Tac alone (Fig. 5,
g and h), implicating the existence of a weak
internalization signal distinct from the LL motif in the cytoplasmic
domain. Taken together, these data suggest that the LL motif in the
cytoplasmic domain of tyrosinase is part of a strong internalization
signal, and secondary signals may also contribute to internalization.
They further imply that tyrosinase may pass through the plasma membrane
at some point during its trafficking.
The Di-leucine Signal of Tyrosinase Interacts with Saturable
Components Involved in Sorting Mediated by Other Di-leucine- and
Tyrosine-based Signals--
By using transient overexpression and a
cell-surface displacement assay, we and others (41, 50, 55, 56) have
shown that conventional sorting signals that conform to a given motif utilize common saturable components to effect localization but that
signals conforming to different motifs utilize distinct saturable components. If the sorting signals in the tyrosinase cytoplasmic domain
corresponded to conventional signals, then it would be expected that
they would compete for binding sites with endogenous proteins bearing
similar signals. Therefore, overexpression of the tyrosinase
cytoplasmic domain would result in mis-sorting and displacement of
these endogenous proteins to the cell surface. We tested this
hypothesis by overexpressing Tac chimeric proteins containing either
well described, conventional sorting signals or the cytoplasmic domain
of tyrosinase and using flow cytometry to analyze the cell-surface
displacement of endogenous or weakly expressed exogenous proteins
containing LL or YXXØ motifs. Levels of expression of each
Tac chimeric protein were monitored by metabolic labeling and
immunoprecipitation, and transfection efficiencies were monitored by
IFM.
HeLa cells were transfected with sufficient levels of plasmid DNA
encoding Tac chimeric proteins to saturate sorting pathways and appear
on the surface of a large fraction of cells. To assay for competition
with LL motifs, cells were co-transfected with low levels of plasmid
DNA encoding the p33 form of human invariant chain (Ip33). Ip33
contains two well characterized LL-like lysosomal targeting signals in
its cytoplasmic domain, DQRDLI and EQLPML
(57-60). As shown in Fig. 6a,
overexpression of TTY caused displacement of Ip33 to the cell surface
at levels comparable to those induced by overexpression of Tac appended
with the LL motif from the CD3
To assay for competition with YXXØ-like sorting signals, we
analyzed transfected cells for the cell-surface appearance of two
endogenous lysosomal membrane proteins, lamp1 and lamp2. Both proteins
rely on well characterized tyrosine-based sorting signals (AGYQTI in lamp1 and AGYEQF
in lamp2) to effect late endosomal localization. As shown in Fig.
7, a and d,
overexpression of TTY results in significant displacement of both
endogenous lamp1 and lamp2 to the cell surface. This displacement is
much greater than that observed upon overexpression of Tac-DKQTLL but
not as substantial as that induced by overexpression of TTL1; this
suggests either a weak affinity for a YXXØ recognition
site, a partial steric block to such a site, or competition for a
distinct and somewhat less limiting factor. Remarkably, disruption of
the LL motif (AA/Y/Y) completely eliminated surface displacement of both lamp1 and lamp2 (Fig. 7, b and e) to levels
comparable to that induced by overexpression of Tac with no signal.
Thus, competition for YXXØ-based sorting is dependent on
the integrity of the tyrosinase LL residues. Disruption of both
tyrosines in the tyrosinase cytoplasmic domain (TTY-LL/A/A) reduced,
but did not eliminate, cell-surface displacement of both lamp1 and
lamp2 (Fig. 7, b and e), suggesting that tyrosine
residues influence the ability of the cytoplasmic domain to compete
with YXXØ signals. However, the same level of displacement
was observed if either tyrosine residue was individually disrupted
(LL/Y/A, LL/A/Y; Fig. 7, c and f). This
suggests either that both tyrosines take part in a novel sorting signal
that weakly or partially competes for cellular factors with
conventional YXXØ motifs or that each tyrosine influences
the context of the LL motif such that it is now able to block access of
cellular components to YXXØ motifs. It further suggests
that HeLa cells retain the ability to distinguish structural features
of sorting signals in the cytoplasmic domain of tyrosinase from other
conventional endosomal sorting signals.
Enlargement of Late Endosomes and Lysosomes by Overexpression of
the Di-leucine Signal of Tyrosinase but Not of CD3 The mechanisms by which proteins are localized to tissue-specific
organelles are poorly understood, but in many cases they may take
advantage of existing redundant pathways to ubiquitous organelles. We
(10) and others (9, 11-13) have previously described evidence that
multiple pathways exist for protein transport to late endosomes and
lysosomes, providing the potential for subversion of one pathway for
localization to tissue-specific late endosomal organelles. Here, we
have extended these studies to demonstrate that a distinct type of
LL-based late endosomal/lysosomal sorting signal exists in the
cytoplasmic domain of tyrosinase, an integral membrane protein
component of the melanosome. The singular behavior of the tyrosinase
sorting signals in non-pigmented cells provides a potential mechanism
by which these signals might be distinguished for localization to the
unique, late endosome-like melanosome in melanocytic cells.
Tyrosinase Localizes to Late Endosomes and Lysosomes of
Non-pigmented Cells--
The cytoplasmic domain of tyrosinase proved
to be both necessary and sufficient for localization to conventional
late endosomes and lysosomes of non-pigmented cells, suggesting that it
contains a classical late endosomal/lysosomal sorting determinant. This was evidenced by nearly complete colocalization of full-length tyrosinase or the TTY chimeric protein with lamp1 and lamp2 in transfected cells, and by predominant cell-surface expression upon
mutagenesis of the LL motif or deletion of the cytoplasmic domain. Our
immunolocalization data extend previous observations in which products
of tyrosinase activity co-fractionated with lysosomes or lysosome-like
structures in transfected cells (Refs. 61-63 and reviewed in Refs. 21
and 23). A requirement for the cytoplasmic domain in proper
localization is consistent with the trafficking defects of the murine
cP allele (30), the OCA phenotype of human
patients with a mutation rendering loss of the cytoplasmic domain (31),
and properties of the cytoplasmic domain of the tyrosinase-related
protein TRP-1 (37).
Like other classical late endosomal sorting signals, the cytoplasmic
domain of tyrosinase also directed internalization and late endosomal
localization from the plasma membrane, since anti-Tac antibody was
internalized and delivered to vesicular compartments in cells
expressing TTY. This is the first described indication that melanosomal
proteins may be internalized and is consistent with previously reported
observations that a fraction of TRP-1 resides at the plasma membrane
(64) and that endocytosed albumin-gold conjugates can be found in
melanosomes (22). If the cytoplasmic domain of tyrosinase functions
similarly in cells of the melanocytic lineage, such recycling might
serve to preserve cellular tyrosinase levels in the face of ongoing
secretion of melanin.
A Second Class of Di-leucine-based Sorting Signals--
A role for
LL-based signals in melanosomal protein localization was first implied
by the loss of intracellular localization of the tyrosinase-related
protein, TRP-1, in fibroblasts and melanocytes upon carboxyl-terminal
truncation that eliminated a sequence overlapping a LL-like signal
(37). In vitro binding of a LL motif from the mouse
tyrosinase cytoplasmic domain to the adaptor-like protein complex, AP3,
lent further indirect support to a critical role for LL signals (38).
Our point mutagenesis studies clearly showed that efficient
internalization and steady state localization of human tyrosinase to
late endosomes and lysosomes is dependent on two consecutive leucine
residues that lie within a sequence conforming to a classical LL
motif (35, 51, 65). Somewhat surprisingly, mutagenesis of either
of two conserved tyrosine residues (Tyr521 or
Tyr525), one of which (Tyr521) lies within a
consensus YXXØ motif, had only minimal effects on steady
state localization and internalization. Furthermore, these residues
were unable to sustain localization of full-length tyrosinase to late
endosomes or lysosomes in the absence of a functional LL motif. These
data show that the LL residues of tyrosinase, but not the tyrosine
residues, are critical components of a sorting determinant for late
endosomes and lysosomes.
The results of an in vivo competition assay suggest that the
critical LL residues form the foundation for a LL motif-based sorting
signal but that this signal interacts either with unique limiting
cellular components or with common LL-binding components in a unique
manner. In this assay, overexpression of TTY resulted in extensive
displacement from intracellular stores to the cell surface of the
invariant chain, a protein that relies on two LL-like motifs for its
sorting to late endosomes (57-59). This displacement was as effective
as that achieved upon overexpression of a chimeric protein containing
the LL signal from CD3
A second indication of the distinctive nature of the tyrosinase sorting
signal came from IFM analysis of overexpressing cells. Overexpression
of TTY or tyrosinase induced enlargement of lamp1-positive late
endosomes and lysosomes in HeLa cells, whereas expression of comparably
high levels of Tac-DKQTLL (with the CD3 Influence of Tyrosine Residues on Context of the Di-leucine
Signal--
Although the tyrosine residues Tyr521 and
Tyr525 do not appear to contribute significantly to signals
with intrinsic sorting activity in HeLa cells, our competition results
suggest that they may contribute to the context in which the LL motif
is recognized. This was evident by the partial reduction in
the ability of overexpressed TTY to displace lamp1 and lamp2 to the
surface upon mutagenesis of either Tyr521,
Tyr525, or both. The partial but not additive effects of
these mutations on lamp displacement indicates that either (i) both
tyrosines are part of a secondary targeting signal, the activity of
which is dependent on the LL motif, or (ii) both tyrosines influence the context of the LL signal such that it can block the interaction of
cellular factors with YXXØ motifs. Since there is no
precedent for either a targeting signal with two critical tyrosines in
the indicated positions or a signal that acts only at an endosomal site
and not at the cell surface, we favor the second model. Regardless of
the mechanism, our data represent the first documented modification of
the properties of an LL sorting motif by a membrane distal sequence and
add to the described effects of membrane proximal residues on LL
sorting efficacy (35, 60, 69-71).
We speculate that the context of the LL motif could be affected by the
tyrosine residues either by an induced conformation that sterically
blocks access to a separate YXXØ-binding site on a putative
LL receptor or to altered specificity for a LL receptor in which
binding sites for LL and YXXØ motifs are spatially closer than in the receptor for the CD3 Implications for Melanosomal Protein Sorting--
We (data not
shown) and others (reviewed in Refs. 21 and 23) have observed
differential localization of lamp1 and tyrosinase in some melanocytes
and melanoma cells, suggesting that melanosomal and late endosomal
integral membrane proteins are differentially recognized by the
melanocyte protein sorting machinery. Our results here provide a
potential mechanism to account for this differential recognition. It is
conceivable that the cellular effectors that interact with the LL
signal from tyrosinase and facilitate sorting via a unique pathway to
late endosomes in HeLa cells may be usurped by cells of the melanocyte
lineage to direct proteins such as tyrosinase and TRP-1 to melanosomes.
Sequence comparison of tyrosinase, TRP-1, and TRP-2 from several
species shows a striking conservation of general structural features of
the cytoplasmic domain that would be consistent with recognition by a
distinct sorting receptor (Fig. 9).
First, each of these proteins retains a LL- (tyrosinase and TRP-1) or
GYXXØ-based (TRP-2) late endosomal/lysosomal sorting signal
at a conserved position close to the membrane. Second, each protein,
particularly tyrosinase, has an unusually high content of basic
residues in the membrane proximal region, amino-terminal to the
lysosomal sorting signal. These residues could interact with acidic
headgroups of phospholipids and/or with a third conserved region of the
cytoplasmic domains rich in acidic amino acid residues and distal to
the conventional sorting signal. If these two regions were to interact
three-dimensionally, a conserved tyrosine residue, analogous to
Tyr525 in human tyrosinase, could be placed in close
proximity to the conventional sorting signal where it might be able to
affect interactions with APs. Although highly speculative, this model
creates a conceptual framework with which to test functions of
different regions of the cytoplasmic domains of tyrosinase, TRP-1, and
TRP-2 in melanosomal protein sorting.
It is particularly intriguing that a determinant capable of mediating
specialized localization in pigmented cells effects localization to
conventional late endosomes and lysosomes in the inappropriate cell
type. The cell type-specific appropriation of a secondary ubiquitous
sorting pathway for a specific localization pattern is well
established. Proteins that are differentially sorted to basolateral and
apical surfaces in polarized cells are delivered to the same
cell-surface domain of fibroblasts in separate vesicles (14-16).
Furthermore, yeast use two pathways for protein delivery to the
vacuole, including one that is dependent on recognition of LL-like
motifs by the homologue of AP3 (1). Thus, further understanding of the
mechanisms of melanosomal protein sorting should provide insights
into general pathways of transport to specialized late endosomal compartments.
by
overexpression studies; overexpression of the tyrosinase signal, but
not the well characterized CD3
signal, induced a 4-fold enlargement of late endosomes and lysosomes and interfered with endosomal sorting
mediated by both tyrosine- and other di-leucine-based signals. These
properties suggest that the tyrosinase and CD3
di-leucine signals
are distinctly recognized and sorted by distinct pathways to late
endosomes in non-pigmented cells. We speculate that melanocytic cells
utilize the second pathway to divert proteins to the melanosome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Wild-type and mutant constructs of
tyrosinase. a, schematic representation of the tyrosinase
gene product showing the predicted signal sequence (SS),
lumenal, transmembrane (TM), and cytoplasmic
(Cyt) domains. The number of amino acid residues in each
domain are indicated at the top. The amino acid sequence of
the predicted wild-type cytoplasmic domain, beginning with the cysteine
residue at position 500, is shown below with putative targeting
determinants shown in bold. The sequence of the
site-directed mutants, named for their amino acid substitution at the
bold sites, are shown below. The transmembrane domain is defined as
ending in the amino acid sequence LVSLL. b, schematic
depiction of the domain structure of Tac/tyrosinase chimeras. The
coding region for the cytoplasmic domain of tyrosinase was appended to
that for the lumenal and transmembrane domains of Tac using a double
PCR technique. The wild-type and mutant amino acid sequences of the
cytoplasmic domains of Tac chimeric constructs are listed below. The
transmembrane domain of Tac is defined here as ending in the amino acid
sequence GLTWQ. c, schematic representation of Tac chimeras
TTL1 and Tac-DKQTLL used in degradation and overexpression studies. The
sequences of characterized tyrosine- and di-leucine-based sorting
signals in the cytoplasmic domains are indicated. The sequence DKQTLL
is derived from the CD3 chain.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PEP7h (39), a rabbit antiserum to the cytoplasmic
domain (gift of V. Hearing, National Institutes of Health, Bethesda), or NY8K (40), a rabbit antiserum to the lumenal domain (gift of L. Old,
Ludwig Institute for Cancer Research, New York). Chimeric proteins
containing the lumenal domain of the interleukin-2 receptor
chain
(Tac) were detected using the mouse monoclonal antibody (mAb) 7G7.B6
(American Type Culture Collection, Rockville, MD) or a rabbit anti-Tac
serum (41). Human invariant chain was detected using mAb LL-1 (gift of
H. Hansen, Immunomedics, Inc., Morris Plains, NJ; Ref. 42) to the
lumenal domain or PIN.1 (gift of P. Cresswell, Yale University, New
Haven, CT; Ref. 43) to the cytoplasmic domain. The following lists
endogenous cellular proteins and reagents used to detect them: lamp1,
mAb H4A3 (Developmental Studies Hybridoma Bank, University of Iowa,
Iowa City) or a rabbit anti-lamp1 serum (gift of M. Fukuda, Scripps
Research Institute, San Diego CA) (44, 45); lamp2, mAb H4B4
(Developmental Studies Hybridoma Bank); transferrin receptor, mAb B3/25
(Boehringer Mannheim); and cation-independent mannose 6-phosphate
receptor, rabbit anti-MPR300 serum (gift of T. Braulke,
University of Göttingen, Germany). FITC, lissamine
rhodamine (LRSC), and phycoerythrin (PE)-conjugated secondary
antibodies were purchased from Jackson ImmunoResearch (West Grove, Pennsylvania).
t3-t2; Ref. 51), all cloned into pCDM8.1, have been previously described.
tail-1 and
tail-2 were made by insertion of a single PCR
amplicon from the original construct. Plasmid DNA was screened for a
unique restriction enzyme digestion pattern relative to the wild type, and sequences of mutants were confirmed by dideoxynucleotide sequencing.
PEP7h (anti-tyrosinase) as described (50), except
that lysates were first normalized for radioactive label content by
trichloroacetic acid precipitation from lysate aliquots.
Immunoprecipitates were fractionated by SDS-PAGE on 10% polyacrylamide
gels, and radioactive bands were quantitated using a Molecular Dynamics
PhosphorImager and ImageQuant software (Sunneyvale, CA). Average
expression per cell was calculated by dividing the total radioactivity
in precipitable bands by the transfection efficiency (fraction of
positively stained cells by immunofluorescence microscopy).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Transient expression of wild-type tyrosinase
in HeLa cells. HeLa cells were transiently transfected with a
plasmid encoding wild-type tyrosinase and fixed 2 days later before
(a) or after (b) treatment for 1 h with 50 µg/ml CHX. Permeabilized cells were analyzed by IFM with a rabbit
antiserum to tyrosinase ( pep7h) and LRSC-conjugated anti-rabbit
Ig.
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Fig. 3.
Colocalization of tyrosinase containing
wild-type and mutant cytoplasmic domains with lamp1 in HeLa cells.
HeLa cells were transiently transfected with plasmids encoding the
indicated tyrosinase with either wild-type (a and
b) or mutated (c-l) cytoplasmic domains and were
analyzed 2 days following transfection by intracellular IFM using a
rabbit antiserum to tyrosinase (left), H4A3 to detect
endogenous lamp1 (right), and FITC- and LRSC-conjugated
secondary antibodies. Prior to fixation, all cells were treated with 50 µg/ml CHX for 1 h at 37 °C. Filled arrowheads
point to examples of vesicles in which both tyrosinase and lamp1 were
detected. Arrows point to examples of vesicles in which only
one protein, but not the other, was stained. Open arrowheads
point to larger vesicles with outer rim staining in cells expressing
tyrosinase with mutations at Tyr525
(g-j).
tail-1 and
tail-2, data not shown) resulted in a drastic
reduction in vesicular staining and the appearance of diffuse staining
throughout the cell with intense staining at cell edges, ruffles, and
spikes, indicating cell-surface expression (Fig. 3c). Many
of the remaining vesicles did not colocalize with lamp1
(arrows, Fig. 3d). Additional mutagenesis of
Tyr525 (AA/Y/A) had a small but consistent effect of
reducing the level of residual vesicular staining remaining after
mutagenesis of the LL motif alone (Fig. 3, k and
l, and data not shown). These results indicate that the LL
motif in the cytoplasmic domain is necessary for efficient sorting of
tyrosinase to late endosomes and lysosomes in HeLa cells. Furthermore,
they suggest that the classical tyrosine-based motif, YHSL, plays no
direct role in sorting to conventional endosomal organelles but that
Tyr525 may be part of a weak secondary sorting signal.
chain)
is a monomeric, cell surface, type I integral membrane protein that we
have previously used as a reporter to characterize a number of
post-Golgi sorting signals (10, 41, 46, 50, 54). A Tac/tyrosinase (TTY)
chimera was generated containing the lumenal and transmembrane domains of Tac and the cytoplasmic domain of tyrosinase (Fig. 1b);
additional Tac chimeric proteins contained the site-directed mutations
of the tyrosinase cytoplasmic domain (Fig. 1b). HeLa cells
were transfected with each of the Tac chimeric constructs, and the
expressed products were detected by IFM. Since the Tac epitopes
detected by our antibodies are sensitive to proteolysis in late
endosomes and lysosomes (46, 50, 51), cells were examined after a 4-h
treatment with leupeptin.
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Fig. 4.
Colocalization of Tac/tyrosinase (TTY)
chimeric proteins with lamp1 in HeLa cells. HeLa cells were
transiently transfected with plasmids encoding the indicated TTY
chimeric proteins with wild-type (a and b) or
mutagenized (c-f) cytoplasmic domains and analyzed 2 days
post-transfection by intracellular IFM using a rabbit anti-Tac
antiserum (left), H4A3 to detect lamp1 (right),
and FITC- and LRSC-conjugated secondary antibodies. Prior to fixation,
all cells were treated with 1 mg/ml leupeptin for 4 h at 37 °C.
Filled arrowheads point to examples of vesicles in which
both the Tac lumenal domain and lamp1 were detected. Arrows
point to examples of vesicles in which only one protein, but not the
other, was stained.
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Fig. 5.
The di-leucine motif of tyrosinase functions
as an internalization signal. HeLa cells were transiently
transfected with plasmids encoding TTY chimeric proteins, Tac, or TTL1
as indicated. Two days later, cells were incubated with 50 µg/ml
purified mAb 7G7.B6 in complete medium at 37 °C, and the antibody
was allowed to internalize for 30 min. Cells were then fixed and
permeabilized, and internalized antibody was detected by IFM using
LRSC-conjugated anti-mouse Ig (Int. 7G7.B6). Transfected
cells were identified by counterstaining permeabilized cells with
rabbit anti-Tac and FITC anti-rabbit Ig (Total Tac chimera).
Note that cells were not treated with leupeptin in this
experiment.
chain (Tac-DKQTLL; Fig.
6b). As previously shown (50), overexpression of Tac
containing no targeting signals (Tac; Fig. 6a) or a
functional YXXØ motif (TTL1; Fig. 6b) did not
induce significant surface expression of Ip33. Mutagenesis of
Tyr521 (TTY-LL/A/Y) did not affect the ability of TTY to
displace Ip33 to the cell surface (Fig. 6c). In contrast,
alteration of the LL signal (TTY-AA/Y/Y) abolished competition to
levels comparable with Tac (Fig. 6c). These data indicate
that the LL motifs in the cytoplasmic domains of tyrosinase and Ip33
use common saturable components to effect localization.
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Fig. 6.
Disruption of sorting mediated by
di-leucine-based motifs by overexpression of TTY. HeLa cells were
transiently transfected with saturating levels (20 µg/10-cm dish) of
plasmids encoding Tac, the indicated Tac chimeras, or a control (Mock)
and low levels (0.5 µg/10 cm dish) of a plasmid encoding Ip33. Three
days after transfection, cells were harvested and stained for surface
expression with a rabbit antibody to Tac, a mouse monoclonal antibody
to invariant chain and then FITC-anti-mouse Ig and PE-anti-rabbit Ig.
Stained cells were analyzed by flow cytometry. Live cells were gated
for high expression of the Tac transgene product ( 5 × 102 units of fluorescence intensity in PE channel in this
experiment) or only for live cells in the Mock sample. Shown is the
intensity of FITC staining for gated cells in a representative of three
experiments. y axis, cell number (normalized for display
purposes); x axis, FITC staining intensity. Parallel
analyses of the same cells by metabolic labeling and
immunoprecipitation and by immunofluorescence microscopy, as described
under "Materials and Methods," showed that all Tac and invariant
chain transgene products were expressed to similar levels in this
experiment. The peak of cells with no surface staining for Ip33 among
transfectants overexpressing TTY, TTY-LL/A/Y, or Tac-DKQTLL is likely
due to cells that were transfected with DNA encoding the Tac chimeric
product but not Ip33, consistent with the relative transfection
efficiencies.
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Fig. 7.
Disruption of sorting mediated by
tyrosine-based motifs by overexpression of TTY. HeLa cells were
transiently transfected with saturating levels (20 µg/10 cm dish) of
plasmids encoding Tac, the indicated Tac chimeras, or a control (not
shown). Three days after transfection, cells were harvested and stained
for surface expression with a rabbit antibody to Tac and mouse
monoclonal antibodies to endogenous lamp1 (a-c) or lamp2
(d-f), and then FITC-anti-mouse Ig and PE-anti-rabbit Ig.
Stained cells were analyzed by flow cytometry. Shown is a
representative of four experiments, and data are presented as in Fig.
6. In the experiment shown, TTY, LL/Y/A, LL/A/Y and LL/A/A were
expressed at comparable levels and about 2-fold higher than Tac,
AA/Y/Y, or Tac-DKQTLL; TTL1 was expressed about half as well as these.
In other experiments with similar results, expression levels for all
constructs were comparable.
--
When cells
overexpressing TTY were analyzed by IFM, the TTY product was observed
to accumulate not only at the cell surface but also in the periphery of
large vesicular structures, the lumen of which was clearly visible
(Fig. 8a). Comparison of lamp1
staining revealed a concomitant enlargement of lamp1-positive
compartments in cells overexpressing TTY relative to untransfected
cells (Fig. 8b). Similarly enlarged lamp1-positive
structures were observed in cells overexpressing full-length tyrosinase
(Fig. 8, i and j). These structures accumulated
more in the perinuclear area than in distal regions of the cell.
Measurement of lamp1-positive structures in untransfected cells or
cells expressing high levels of TTY at the cell surface revealed a
2-fold increase in diameter in the latter cells (1.026-µm median
diameter), representing a 4-fold increase in surface area, relative to
the untransfected cells (0.498 µm median diameter; see Table
I). This difference was statistically
significant (p < 0.0001). Enlargement of
lamp1-positive compartments was also observed upon overexpression of
TTY-LL/A/A (Fig. 8, e and f), TTY-LL/Y/A and
TTY-LL/A/Y (not shown), but not of TTY-AA/Y/Y (Fig. 8, c and
d) or tyrosinase-AA/Y/Y (Fig. 8, k and
l), indicating that the enlargement was dependent on the
integrity of the LL motif but not of either or both tyrosine residues.
Importantly, overexpression of comparably high levels of Tac-DKQTLL,
with the LL motif from CD3
, failed to induce a dramatic enlargement
of lamp1-positive structures (0.573 µm median diameter), although
there was a statistically significant difference in comparison to
untransfected cells (0.498 µm median diameter; p < 0.0001) (Fig. 8, g and h, and Table I). The
massive enlargement observed upon overexpression of TTY is likely due
to disruption of sorting of a protein(s) required for maintenance of
late endosomal volume or surface area. These results suggest that the
LL signal of tyrosinase mediates sorting through a common pathway with
such a protein, whereas the LL signal of CD3
does not. We conclude, therefore, that these two signals direct sorting to late endosomes via
distinct pathways.
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Fig. 8.
Overexpression of the tyrosinase cytoplasmic
domain induces an enlargement of lamp1-positive vesicular
structures. HeLa cells were transfected with saturating levels (8 µg/60-mm dish) of plasmid encoding TTY (a and
b), TTY-AA/Y/Y (c and d), TTY-LL/A/A
(e and f), Tac-DKQTLL (g and
h), tyrosinase (i and j), or
tyrosinase-AA/Y/Y (k and l). Three days after
transfection, coverslips were fixed and analyzed by IFM using
antibodies to Tac (a, c, e, and g) or tyrosinase
(i and k) and lamp1 (right panels).
All cells are shown at the same level of magnification.
Inset shows a higher magnification of lamp1 staining of Tac
positive cells to emphasize the relative sizes of the lamp1-positive
compartments. Transfection efficiencies were monitored by counting the
fraction of Tac expressing cells, and aliquots of cells were also
subjected to metabolic pulse labeling and immunoprecipitation to
determine expression levels. Expression per cell, determined as
described under "Materials and Methods," was comparable among all
Tac chimeric samples (relative expression: TTY, 1.0; Tac-DKQTLL, 1.04;
TTY-AA/Y/Y, 1.38; TTY-LL/A/A, 1.34) and among both tyrosinase samples
(relative expression: tyrosinase, 1.0; tyrosinase-AA/Y/Y, 2.88).
Increased diameter of lamp1-positive compartments in cells
overexpressing the tyrosinase cytoplasmic domain
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(51) and was much greater than that observed
with the YXXØ signal from lamp1 as previously shown (50).
We conclude from these results that TTY effectively competes for
limiting cellular factors in sorting and internalization mediated by
well characterized LL motifs. However, overexpression of TTY also
resulted in significant cell-surface displacement of lamp1 and lamp2,
two proteins that rely on YXXØ signals for lysosomal
sorting and internalization. This displacement was not as great as that
induced by overexpression of the lamp1 YXXØ signal, but was
much greater than that induced by overexpression of Tac alone or of the
LL signal from CD3
. We interpret this displacement as an indication
that TTY partially competes for limiting cellular factors involved in
either the recognition of YXXØ signals or sorting and
internalization mediated by these signals. Importantly, induction of
lamp1 and lamp2 surface expression was ablated by mutagenesis of the LL
residues, indicating that competition for YXXØ signals was
dependent on the integrity of the LL motif. This interaction with
YXXØ-based recognition/sorting factors represents a novel
property of a LL-based sorting signal, since competition for
YXXØ motifs is not observed with the CD3
LL signal (here
and Ref. 50). The data therefore suggest that the tyrosinase LL-based
signal interacts with at least partially distinct factors to direct
sorting to late endosomes and lysosomes in HeLa cells.
LL signal) had no such
effect. This effect was dependent on the LL residues of the tyrosinase
cytoplasmic domain but not the distal tyrosine residues. The
enlargement could be due to either accumulation of lumenal contents
(failure to degrade lumenal contents), disregulation of ion transport
across the late endosomal membrane (increased lumenal ionic strength to
cause swelling), or failure to form vesicles from the limiting membrane
(inhibition of budding), any of which most likely results from
competitive inhibition of a specific sorting step for a protein
critical in each process by overexpression of the tyrosinase, but not
the CD3
, LL signal. Interestingly, similar observations have been
made upon overexpression of the invariant chain (66, 67). The sequence
of the membrane proximal of the two LL-like signals in invariant chain
is similar to that of tyrosinase (57, 59, 60, 68), suggesting that both
proteins may be sorted by similar mechanisms.
LL motif. Distinct receptors for
each LL motif might provide a mechanism by which their differential sorting can be effected, consistent with the morphological
characteristics of the TTY- and tyrosinase-overexpressing cells.
Potential candidates for such LL receptors would be the adaptor
complexes AP3, AP2, and AP1; the LL motifs of mouse tyrosinase and
CD3
have been described to bind, respectively, to AP3 and AP2/AP1
(38, 72). The affinity of a given signal for different AP complexes has been proposed to regulate sorting at different sites within the endosomal pathway (25, 34, 36, 73, 74); thus, we would predict that
differential binding, influenced by the tyrosine residues, would result
in distinct localization pathways for similar steady state
distributions of these two proteins.
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Fig. 9.
Sequence alignment of the cytoplasmic domains
of tyrosinase and tyrosinase-related proteins from different
species. Shown are the deduced amino acid sequences of the
predicted cytoplasmic domains from the indicated proteins, aligned into
regions as defined in the text. Basic residues within the membrane
proximal region are indicated in bold; consensus sorting
signals are underlined; acidic residues in the distal
region are shaded, and the conserved tyrosine residue is
boxed. Sequences were obtained from the
GenBankTM data base.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Drs. V. Hearing (National Institutes of Health, Bethesda), W. Storkus (University of Pittsburgh), and S. Topalian (National Institutes of Health) for generously providing reagents and guidance for analysis of tyrosinase. We also thank Drs. M. Fukuda (Scripps Institute, La Jolla, CA), L. Old (Ludwig Institute, New York), H. Hansen (Immunomedics, Morris Plains, NJ), and T. Braulke (University of Göttingen, Göttingen, Germany) for providing necessary reagents; Dr. D. Roof (University of Pennsylvania) for use of the microscope facility; Dr. C. Deutsch (University of Pennsylvania) for assistance with statistical analyses; and Dr. W. Skach (University of Pennsylvania), Dr. M. Birnbaum (University of Pennsylvania), Dr. J. Keen (Thomas Jefferson University, Philadelphia), and Shaheen Sutterwala for critical review of the manuscript.
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Note Added in Proof |
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During review of this manuscript, Simmen et al. (Simmon, T., Schmidt, A., Hunziker, W., and Beermann. F. (1999) J. Cell Sci. 112, 45-53) published a characterization of cytoplasmic domain signals required for lysosomal targeting of murine tyrosinase in transfected MDCK cells. They found a similar requirement for the LL motif. However, their mutational analysis (but not their deletion analysis) implicated the proximal tyrosine residue as also necessary for targeting. We feel that the data are consistant with our interpretation of a role for the tyrosine residue in regulating the conformation of the LL motif; mouse tyrosinase contains an additional leucine residue following the YXXØ motif (the sequence is YHSLL), and we speculate that mutagenesis of the tyrosine results in an inappropriate hydrophobic interaction of these two leucine residues with the bona fide LL motif, resulting in its functional inactivation.
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FOOTNOTES |
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* This work was supported in part by Grant RPG 97-003-01-BE from the American Cancer Society and Grants R21 AI 42055 and R21 AI 42617 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported in part by a National Research Service Award
1F32-AI09946 and by Training Grant T32-CA09671 from the National
Institutes of Health.
§ Supported in part by Training Grant T32-EY07131-06 from the National Institutes of Health.
¶ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 277 John Morgan Bldg./6082, 36th and Hamilton Walk, Philadelphia, PA 19104-6082. Tel.: 215-898-3204; Fax: 215-573-4345; E-mail: marksm{at}mail.med.upenn.edu.
2 J. F. Berson, D. W. Frank, P. A. Calvo, B. M. Bieler, and M. S. Marks, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: OCA, oculocutaneous albinism; Ø, amino acid with bulky hydrophobic side chain; LL, di-leucine; YXXØ, tyrosine-based motif; AP, adaptor-like complex; TRP, tyrosinase-related protein; mAb, monoclonal antibody; IFM, immunofluorescence microscopy; CHX, cycloheximide; FITC, fluorescein isothiocyanate; LRSC, lissamine rhodamine-sulfonyl chloride; PE, phycoerythrin; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ER, endoplasmic reticulum.
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