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First published online 18 April 2006
doi: 10.1242/jcs.02930

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An unconventional dileucine-based motif and a novel cytosolic motif are required for the lysosomal and melanosomal targeting of OA1

¹ San Raffalele Scientific Institute, DIBIT, Via Olgettina 58, 20132 Milan, Italy
² Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, University of Lecce, Via Provinciale Lecce-Monteroni, 73100 Lecce, Italy
³ Department of Experimental Medicine, University of Genova Medical School, Via deToni 14, 16132 Genoa, Italy

View larger version (40K): [in a new window]   Fig. 1. OA1 is targeted to lysosomes in HeLa cells and to lysosomes and melanosomes in MNT1 cells. (A) Immunofluorescence analysis of HeLa cells transiently transfected with an expression vector for wild-type mycHis-tagged OA1. Exogenous OA1 was immunodecorated with anti-His6 antibodies and lysosomes were labeled using the fluid-phase marker Lucifer Yellow by chasing for 1 hour prior to fixation (L.Y.). In the merge, areas of colocalization can be observed as yellow/orange. Bar, 10 µm. (B) Quantitative immunofluorescence analysis of the colocalization between endogenous OA1 and markers of various intracellular compartments in MNT1 cells. Cells were double labeled with anti-OA1 antibodies and with antibodies to LAMP1, Pmel17, TRP1, EEA1, or with Lucifer Yellow at 1 hour chase. Results are expressed as the percentage of double-positive vesicles out of all OA1-positive compartments per cell and represent the mean ± s.d. of the data pooled from one or two independent experiments. The number of vesicles and cells counted is indicated.

View larger version (44K): [in a new window]   Fig. 2. The third cytosolic loop (i3) and C-terminal tail (CT) of OA1 contain lysosomal sorting signals. (A) Schematic representation of the predicted heptahelical topology of OA1, as supported by previous bioinformatic, biochemical and morphological studies (Schiaffino et al., 1999). The position of transmembrane domains (I-VII), hydrophilic lumenal (e1-e3) and cytosolic (i1-i3) loops, and the C-terminal tail (CT) is indicated. The portions of the protein utilized to generate LAMP/OA1 chimeras are depicted as black circles, and missense mutations previously described in i3 are shown. The black bar in i3 delimits the 19 N-terminal amino acids of the third cytosolic loop, named i3FY and containing the dileucine-like motif. Between the round brackets is the `S-Y' sequence, which is deleted in the OA1/SY mutant. The black bar in CT separates the 25 N-terminal amino acids of the CT, named CT2 and containing the `WE' motif, from the 65 C-terminal amino acids of the CT, named CTCT2 and deleted in the OA1CT2 mutant. Arrowheads indicate the LL or WE residues, which represent the core of the corresponding motifs and are mutated to AA in the OA1/LLAA or OA1/WEAA mutants, respectively. (B) Immunofluorescence analysis of HeLa cells transiently transfected with expression vectors for LAMP/OA1 chimeras and immunodecorated with anti-rat LAMP1 antibodies. Cells were cotransfected with an expression vector for farnesylated GFP to label the plasma membrane (EGFP-F). In the merge, areas of colocalization can be observed in yellow/orange. Distribution patterns of recombinant proteins are indicated on the right. LAMP/i2 behaves as LAMP/i1 (not shown). Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 3. The dileucine-like motif in i3 functions as a signal sufficient for lysosomal sorting in HeLa cells. (A) Structure, sequence and cross-species conservation of the LAMP/i3FY construct, carrying the 19 N-terminal amino acids of i3, and of its deletion and mutagenesis variants. N-term, N-terminus of rat LAMP1; TM, transmembrane domain of rat LAMP1; these were fused with the cytosolic loop of OA1. (B) Immunofluorescence analysis of HeLa cells transiently transfected with expression vectors for LAMP/i3 chimeras and immunodecorated with anti-rat LAMP1 antibodies. Cells were counterstained with the fluid-phase marker Lucifer Yellow at 1 hour chase prior to fixation to label the lysosomes (L.Y.), or were cotransfected with an expression vector for farnesylated GFP to label the plasma membrane (EGFP-F). In the merge, areas of colocalization can be observed in yellow/orange. LAMP/i3FG behaves as LAMP/i3FY LLAA (not shown). LAMP/i3FY is a LAMP/OA1 chimera carrying the 18 C-terminal amino acids of i3. Bar, 10 µm.

View larger version (74K): [in a new window]   Fig. 4. The WE motif in CT functions as a signal sufficient for lysosomal sorting in HeLa cells. (A) Structure, sequence and cross-species conservation of the LAMP/CT2 construct, carrying the 25 N-terminal amino acids of CT, and of its deletion and mutagenesis variants. N-term, N-terminus of rat LAMP1; TM, transmembrane domain of rat LAMP1; these were fused with the CT of OA1. (B) Immunofluorescence analysis of HeLa cells transiently transfected with expression vectors for LAMP/CT chimeras and processed as in Fig. 3B. In the merge, areas of colocalization can be observed in yellow/orange. LAMP/CT2M11 behaves as LAMP/CT2M5 WEAA (not shown). LAMP/CTCT2 is a LAMP/OA1 chimera carrying the 65 C-terminal amino acids of CT. Bar, 10 µm.

View larger version (31K): [in a new window]   Fig. 5. The dileucine-like and WE motifs are sufficient for lysosomal/melanosomal targeting in MNT1 cells. (A) Quantitative immunofluorescence analysis of the subcellular distribution displayed by LAMP/OA1 chimeras in transiently transfected MNT1 cells. Following immunodecoration with anti-rat LAMP1 antibodies, transfected cells were counted and classified into three categories, based on the vesicular (VE), plasma membrane (PM) or mixed (MX) distribution of the recombinant proteins (see Materials and Methods for details). Results are expressed as the percentage of cells showing a specific distribution pattern and represent the mean ± s.e.m. of 3-5 independent experiments. The total number of cells counted is indicated. (B) Quantitative immunofluorescence analysis of the colocalization between LAMP/OA1 chimeras and endogenous OA1 in transiently transfected MNT1 cells. Cells were double labeled with anti-rat LAMP1 and anti-OA1 antibodies. Results are expressed as the percentage of double-positive vesicles out of all rat LAMP1-positive compartments per cell and represent the mean ± s.d. of the data pooled from 2-5 independent experiments. The number of vesicles and cells counted is indicated. No colocalization differences were noted between cells displaying vesicular or mixed distribution patterns of the recombinant proteins, thus all data concerning each chimera were pooled together.

View larger version (61K): [in a new window]   Fig. 6. The dileucine-like and WE motifs are necessary for intracellular targeting of OA1 in HeLa cells. (A) Immunofluorescence analysis of HeLa cells transiently transfected with expression vectors for mycHis-tagged OA1 wild type (OA1 WT), single mutant (OA1 SY and OA1 WEAA) or double mutant (OA1 SY/WEAA) and immunodecorated with anti-His6 antibodies. OA1 SY/WEAA was colocalized by confocal microscopy with co-expressed farnesylated GFP (EGFP-F), which labels the plasma membrane. In the merge, areas of colocalization can be observed in yellow/orange. Bar, 10 µm. (B) Quantitative immunofluorescence analysis of the subcellular distribution displayed by full-length OA1 constructs in transiently transfected HeLa cells. Following immunodecoration with anti-His6 antibodies, transfected cells were counted and classified as described in Fig. 5A. Results represent the mean ± s.e.m. of 5-10 independent experiments. The total number of cells counted is indicated.

View larger version (50K): [in a new window]   Fig. 7. The dileucine-like and WE motifs are necessary for intracellular targeting of OA1 in MNT1 cells. (A) Immunofluorescence analysis of MNT1 cells transiently transfected with expression vectors for mycHis-tagged OA1 wild-type (OA1 WT), single mutant (OA1 SY and OA1/WEAA) or double mutant (OA1 SY/WEAA) and immunodecorated with anti-His6 antibodies. OA1 SY/WEAA was colocalized by confocal microscopy with endogenous Na+/K+ ATPase, a marker of the plasma membrane. In the merge, areas of colocalization can be observed in yellow/orange. Bar, 10 µm. (B) Quantitative immunofluorescence analysis of the subcellular distribution displayed by full-length OA1 constructs in transiently transfected MNT1 cells. Following immunodecoration with anti-His6 antibodies, transfected cells were counted and classified as described in Fig. 5A. Results represent the mean ± s.e.m. of 3-10 independent experiments. The total number of cells counted is indicated.

View larger version (36K): [in a new window]   Fig. 8. The distal portion of the C-terminal tail is not necessary for lysosomal targeting of OA1 in HeLa cells. (A) Immunofluorescence analysis of HeLa cells transiently transfected with an expression vector for mycHis-tagged OA1CT2, which lacks the 65 C-terminal amino acids of CT. Exogenous OA1 was immunodecorated with anti-His6 antibodies and lysosomes were labeled using the fluid-phase marker Lucifer Yellow at 1 hour chase prior to fixation (L.Y.). In the merge, areas of colocalization can be observed in yellow/orange. Bar, 10 µm. (B) Quantitative immunofluorescence analysis of the subcellular distribution displayed by OA1CT2 constructs in transiently transfected HeLa cells. Following immunodecoration with anti-His6 antibodies, transfected cells were counted and classified as described in Fig. 5A. Results represent the mean ± s.e.m. of 3-8 independent experiments. The total number of cells counted is indicated.

View larger version (25K): [in a new window]   Fig. 9. The distal portion of the C-terminal tail is not necessary for lysosomal/melanosomal targeting of OA1 in MNT1 cells. (A) Quantitative immunofluorescence analysis of the colocalization between exogenous mycHis-tagged OA1CT2 and markers of various intracellular compartments in transiently transfected MNT1 cells. Cells were double labeled with anti-His6 antibodies and with antibodies to OA1 (that recognize the endogenous protein, but not OA1CT2), LAMP1, Pmel17, EEA1, or with Lucifer Yellow at 1 hour chase. Results are expressed as the percentage of double-positive vesicles out of all His6-positive compartments per cell and represent the mean ± s.d. of the data pooled from 1-6 independent experiments. The number of vesicles and cells counted is indicated. (B) Quantitative immunofluorescence analysis of the subcellular distribution displayed by OA1CT2 constructs in transiently transfected MNT1 cells. Following immunodecoration with anti-His6 antibodies, transfected cells were counted and classified as described in Fig. 5A. Results represent the mean ± s.e.m. of 3-10 independent experiments. The total number of cells counted is indicated.