A dileucine motif and a cluster of acidic amino acids in the second cytoplasmic domain of the batten disease-related CLN3 protein are required for efficient lysosomal targeting
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A dileucine motif and a cluster of acidic amino acids in the second cytoplasmic domain of the batten disease-related CLN3 protein are required for efficient lysosomal targeting. / Storch, Stephan; Pohl, Sandra; Braulke, Thomas.
In: J BIOL CHEM, Vol. 279, No. 51, 17.12.2004, p. 53625-34.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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T1 - A dileucine motif and a cluster of acidic amino acids in the second cytoplasmic domain of the batten disease-related CLN3 protein are required for efficient lysosomal targeting
AU - Storch, Stephan
AU - Pohl, Sandra
AU - Braulke, Thomas
PY - 2004/12/17
Y1 - 2004/12/17
N2 - The juvenile form of ceroid lipofuscinosis (Batten disease) is a neurodegenerative lysosomal storage disorder caused by mutations in the CLN3 gene. CLN3 encodes a multimembrane-spanning protein of unknown function, which is mainly localized in lysosomes in non-neuronal cells and in endosomes in neuronal cells. For this study we constructed chimeric proteins of three CLN3 cytoplasmic domains fused to the lumenal and transmembrane domains of the reporter proteins LAMP-1 and lysosomal acid phosphatase to identify lysosomal targeting motifs and to determine the intracellular transport and subcellular localization of the chimera in transfected cell lines. We report that a novel type of dileucine-based sorting motif, EEEX(8)LI, present in the second cytoplasmic domain of CLN3, is sufficient for proper targeting to lysosomes. The first cytoplasmic domain of CLN3 and the mutation of the dileucine motif resulted in a partial missorting of chimeric proteins to the plasma membrane. At equilibrium, 4-13% of the different chimera are present at the cell surface. Analysis of lysosome-specific proteolytic processing revealed that lysosomal acid phosphatase chimera containing the second cytoplasmic domain of CLN3 showed the highest rate of lysosomal delivery, whereas the C terminus of CLN3 was found to be less efficient in lysosomal targeting. However, none of these cytosolic CLN3 domains was able to interact with AP-1, AP-3, or GGA3 adaptor complexes. These data revealed that lysosomal sorting motifs located in an intramolecular cytoplasmic domain of a multimembrane-spanning protein have different structural requirements for adaptor binding than sorting signals found in the C-terminal cytoplasmic domains of single- or dual-spanning lysosomal membrane proteins.
AB - The juvenile form of ceroid lipofuscinosis (Batten disease) is a neurodegenerative lysosomal storage disorder caused by mutations in the CLN3 gene. CLN3 encodes a multimembrane-spanning protein of unknown function, which is mainly localized in lysosomes in non-neuronal cells and in endosomes in neuronal cells. For this study we constructed chimeric proteins of three CLN3 cytoplasmic domains fused to the lumenal and transmembrane domains of the reporter proteins LAMP-1 and lysosomal acid phosphatase to identify lysosomal targeting motifs and to determine the intracellular transport and subcellular localization of the chimera in transfected cell lines. We report that a novel type of dileucine-based sorting motif, EEEX(8)LI, present in the second cytoplasmic domain of CLN3, is sufficient for proper targeting to lysosomes. The first cytoplasmic domain of CLN3 and the mutation of the dileucine motif resulted in a partial missorting of chimeric proteins to the plasma membrane. At equilibrium, 4-13% of the different chimera are present at the cell surface. Analysis of lysosome-specific proteolytic processing revealed that lysosomal acid phosphatase chimera containing the second cytoplasmic domain of CLN3 showed the highest rate of lysosomal delivery, whereas the C terminus of CLN3 was found to be less efficient in lysosomal targeting. However, none of these cytosolic CLN3 domains was able to interact with AP-1, AP-3, or GGA3 adaptor complexes. These data revealed that lysosomal sorting motifs located in an intramolecular cytoplasmic domain of a multimembrane-spanning protein have different structural requirements for adaptor binding than sorting signals found in the C-terminal cytoplasmic domains of single- or dual-spanning lysosomal membrane proteins.
KW - Acid Phosphatase
KW - Amino Acid Motifs
KW - Amino Acid Sequence
KW - Amino Acids
KW - Animals
KW - Antigens, CD
KW - Biotinylation
KW - Blotting, Western
KW - Brain
KW - CHO Cells
KW - Cell Membrane
KW - Cricetinae
KW - Cytoplasm
KW - Cytosol
KW - DNA, Complementary
KW - Genetic Vectors
KW - Glutathione Transferase
KW - HeLa Cells
KW - Humans
KW - Immunoprecipitation
KW - Leucine
KW - Lysosome-Associated Membrane Glycoproteins
KW - Lysosomes
KW - Membrane Glycoproteins
KW - Microscopy, Fluorescence
KW - Molecular Chaperones
KW - Molecular Sequence Data
KW - Protein Structure, Tertiary
KW - Protein Transport
KW - Recombinant Fusion Proteins
KW - Transfection
U2 - 10.1074/jbc.M410930200
DO - 10.1074/jbc.M410930200
M3 - SCORING: Journal article
C2 - 15469932
VL - 279
SP - 53625
EP - 53634
JO - J BIOL CHEM
JF - J BIOL CHEM
SN - 0021-9258
IS - 51
ER -