The AP-3-dependent targeting of the melanosomal glycoprotein QNR-71 requires a di-leucine-based sorting signal
Roland Le Borgne*,
Nathalie Planque
,
Patrick Martin,
Frédérique Dewitte,
Simon Saule and
Bernard Hoflack
,¶
Institut de Biologie de Lille, CNRS EP525, Institut Pasteur de Lille, BP
447, 59021 Lille cedex, France
*
Present address: Ecole Normale Supérieure,
CNRS équipe ATIPE/UMR 8544, 46, rue d'Ulm,
75230 Paris cedex 05, France
Present address: UMR 146, Institut Curie- Section de recherche, Centre
Universitaire, Bâtiment 110, 91405 Orsay Cedex,
France
Present address: UMR 144, Institut Curie, 26, rue d'Ulm, 75248 Paris cedex 5,
France
¶
Author for correspondence (e-mail:
bernard.hoflack{at}curie.fr
)
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Fig. 7. AP-3 recruitment and expression of QNR-71 mutants. HeLa cells were infected
and transfected as indicated in the legend of
Fig. 6 with plasmids encoding
the L551G (a,b) or Y514A (c,d) mutated forms of VSV-G-tagged QNR-71. After 3
hours of expression, the cells were fixed and processed for indirect
immunofluorescence and labeled with the P5D4 monoclonal anti VSV-G antibody
followed by a FITC-conjugated donkey anti-mouse antibody (a,c). AP-3 was
detected using the polyclonal antibody against the  -subunit followed by
a Texas Red-conjugated donkey anti-rabbit antibody (b,d). Non-transfected
cells are labeled with an asterisk.
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Fig. 1. Wild-type and mutant VSV-G epitope-tagged QNR-71 constructs used in the
study. Schematic representation of the QNR-71 gene product showing the
predicted signal sequence (SS), lumenal, transmembrane (TMD) and cytoplasmic
domains. The number of amino acids in each domain is indicated. The potential
sorting signals in the predicted cytoplasmic domain are shown in bold. The
VSV-G epitope is located at the N terminus of the gene product immediately
downstream of the signal sequence. The gpI-QNR-71 chimeric protein is composed
of the lumenal and transmembrane domains of the VZV envelope glycoprotein gpI
fused to a part of the QNR-71 tail (amino acids 537 to 556) containing a
di-leucine motif.
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Fig. 2. Localization and half life of QNR-71 in cells from the quail retinal
pigmented epithelium. VSV-G-tagged QNR-71 was transiently expressed in quail
cells dissociated from the retinal pigmented epithelium at E8. The cells were
then fixed and processed for immunofluorescence using the monoclonal P5D4
anti-VSV-G antibody (a,d). (b) The phase contrast image of a, where pigment
granules appear as black rod structures. In (c), pigmented quail or HeLa cells
were grown on 24-well dishes and transfected with DNAs encoding VSV-G-epitope
tagged QNR-71 as indicated in Materials and Methods. The cells were then pulse
labeled for 30 minutes with [35S]methionine/cysteine and chased for
the indicated period of time. QNR-71 was immunoprecipitated with the
anti-VSV-G antibody, and analyzed by SDS-PAGE. The position of the precursor
(P) and mature (M) forms of the immunoprecipitated proteins are indicated. (d)
Cells were first treated with 10 mM NH4Cl for 1 hour before
fixation. (d) is the superimposition of phase contrast and fluorescence
images. Some of the large vacuoles contain both QNR-71 and melanin
pigments.
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Fig. 3. Localization of QNR-71 in HeLa cells. HeLa cells were transiently
transfected with plasmids encoding the VSV-G tagged QNR-71 and were fixed 48
hours after transfection. They were then processed for indirect
immunofluorescence and stained using a polyclonal (a,c) or the monoclonal (e)
anti-VSV-G antibody together with the monoclonal anti-EEA1 antibody (b), the
monoclonal anti-LampI antibody (d), or a polyclonal anti-Man 6-P/IGF II
receptor antibody (f). QNR-71 was detected using FITC-conjugated secondary
antibody, while EEA1, Lamp I and Man 6-P/IGF II receptor were decorated using
Texas Red-coupled secondary antibodies. Overlaid images are shown on the
right.
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Fig. 8. Quantitation of membrane bound AP-3 in HeLa cells overexpressing the
different QNR-71 constructs. The intensity of the fluorescent signals
corresponding to the -subunit of AP-3 as shown in Figs
5,
6 was quantitated from 75 to
100 non-transfected (MOCK) cells or cells overexpressing the wild-type (WT) or
the mutated (L551G, L552G, Y514A) versions of VSV-G-tagged QNR-71 and analyzed
as indicated in Materials and Methods. The values represent the
means±s.d. of four different experiments.
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Fig. 4. Localization of wild-type and mutant QNR-71. The wild-type QNR-71 (a,b),
and L552G (c,d) and Y514A (e,f) mutated versions of VSV-G epitope-tagged
QNR-71 were transiently transfected in HeLa cells (a,c,e) or pigmented RPE
quail cells (b,d,f). Two days after transfection, cells were fixed and
processed for immunofluorescence to detect the QNR-71 using the monoclonal
anti-VSV-G antibody.
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Fig. 5. The di-leucine-based sorting signal of QNR-71 is necessary and sufficient
for its targeting. The gpI-QNR-71 chimera was transiently expressed together
with the wild-type VSV-G-QNR-71 in HeLa cells (a,b) or alone (c). Two days
after transfection, cells were fixed, processed for immunofluorescence and
stained using a polyclonal anti-gpI antibody (a,c), together with the
monoclonal anti-VSV-G antibody (b), or the monoclonal anti-EEA1 antibody
(d).
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Fig. 9. AP-3 dependent transport of QNR-71. HeLa cells transfected with the
wild-type VSV-G-QNR-71 construct were grown on glass coverslips. They were
either left untreated (a,d) or treated with sense (b,e) or antisense (c,f)
oligonucleotides. After 48 hours, antibodies directed against LampI (d-f) and
VSV-G (a-c) were added to cell culture medium and allowed to be internalized
for 4 hours at 37°C. After extensive washes, the cells were fixed and the
internalized antibodies were detected using fluorescently labeled secondary
antibodies. Asterisks indicate untransfected cells.
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Fig. 10. Cytoplasmic domains of melanosomal, lysosomal and vacuolar transmembrane
glycoproteins. Sequence alignment of the cytoplasmic domain of integral
membrane proteins targeted to lysosomes/vacuole or melanosomes. Boxed are the
di-leucine- and tyrosine-based sorting signals from the indicated proteins.
The indicated numbers reflect the number of amino acids between two domains or
two signals. The most conserved residues are indicated in bold. Sequences were
obtained from GenBank.
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© The Company of Biologists Ltd 2001
-adaptin (b) or the polyclonal
anti-