Identification of nuclear localisation sequences in spastin (SPG4) using a novel Tetra-GFP reporter system.
Mutations in the human spastin gene (SPG4) cause the most prevalent form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterised by progressive weakness and spasticity of the lower limbs. We address the question of intracellular localisation of spastin. Using polyclonal antibodies against N-terminal spastin sequences, we find that the native protein is localised in both the perinuclear cytoplasm and the nucleus. To identify structural motifs within the protein that can explain entry into the nucleus, we developed a reporter system to test nuclear localisation sequence (NLS)-functionality based on four in-frame fused copies of green fluorescent protein. Using this novel tool we demonstrate that spastin carries two NLSs located in exons 1 and 6. Both are independently functional in mediating nuclear entry.
Beetz
Christian
C
Institut für Pathologie, Klinikum der Friedrich-Schiller Universität Jena, Ziegelmühlenweg 1, 07740 Jena, Germany.
Brodhun
Michael
M
Moutzouris
Konstantin
K
Kiehntopf
Michael
M
Berndt
Alexander
A
Lehnert
Dirk
D
Deufel
Thomas
T
Bastmeyer
Martin
M
Schickel
Jörg
J
D000251:Adenosine Triphosphatases; D020816:Amino Acid Motifs; D000906:Antibodies / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000074181:Spastin; D014162:Transfection
D000906:Antibodies; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D000074181:Spastin; C400283:SPAST protein, human