Abstract
Despite thorough structure-function analyses, it remains unclear how CapG, a ubiquitous F-actin barbed end capping protein that controls actin microfilament turnover in cells, is able to reside in the nucleus and cytoplasm, whereas structurally related actin-binding proteins are predominantly cytoplasmic. Here we report the molecular basis for the different subcellular localization of CapG, severin, and fragminP. Green fluorescent protein-tagged fragminP and severin accumulate in the nucleus upon treatment of transfected cells with the CRM1 inhibitor leptomycin B. We identified a nuclear export sequence in severin and fragminP, which is absent in CapG. Deletion of amino acids Met(1)-Leu(27) resulted in nuclear accumulation of severin and fragminP. Tagging this sequence to CapG triggered nuclear export, whereas mutation of single leucine residues (Leu(17), Leu(21), and Leu(27)) in the export sequence inhibited nuclear export. Based on these findings, a nuclear export signal was identified in myopodin, a muscle-specific actin-binding protein, and the Bloom syndrome protein, a RecQ-like helicase. Deletion of the myopodin nuclear export sequence blocked invasion into collagen type I of C2C12 cells transiently overexpressing myopodin. Our findings explain regulated subcellular targeting of distinct classes of actin-binding proteins.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / metabolism
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Active Transport, Cell Nucleus
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Adenosine Triphosphatases / chemistry
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Amino Acid Sequence
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Animals
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Antibiotics, Antineoplastic / pharmacology
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Cell Line
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Cell Nucleus / metabolism*
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Cloning, Molecular
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Collagen / metabolism
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Cytoplasm / metabolism*
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DNA Helicases / chemistry
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DNA, Complementary / metabolism
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Dalteparin / chemistry
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Dogs
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / metabolism
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Fatty Acids, Unsaturated / pharmacology
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Gene Deletion
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Green Fluorescent Proteins
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Humans
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Immunohistochemistry
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Leucine / chemistry
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Luminescent Proteins / metabolism
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Methionine / chemistry
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Microfilament Proteins / chemistry*
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Microfilament Proteins / physiology*
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Microscopy, Fluorescence
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Molecular Sequence Data
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Muscles / metabolism
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Mutation
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Nuclear Proteins / chemistry*
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Nuclear Proteins / physiology*
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Protein Binding
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Protein Structure, Tertiary
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Protozoan Proteins / chemistry
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RecQ Helicases
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / metabolism
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Time Factors
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Transfection
Substances
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Actins
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Antibiotics, Antineoplastic
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DNA, Complementary
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Fatty Acids, Unsaturated
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Luminescent Proteins
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Microfilament Proteins
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Nuclear Proteins
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Protozoan Proteins
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Recombinant Fusion Proteins
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Recombinant Proteins
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SYNPO2 protein, human
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severin protein, Dictyostelium
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Green Fluorescent Proteins
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CAPG protein, human
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Collagen
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Methionine
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Adenosine Triphosphatases
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Bloom syndrome protein
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DNA Helicases
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RecQ Helicases
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Leucine
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Dalteparin
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leptomycin B