Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles.
Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network.
Holleran
E A
EA
Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia 19104, USA.
Tokito
M K
MK
Karki
S
S
Holzbaur
E L
EL
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015415:Biomarkers; D002460:Cell Line / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000072159:Dynactin Complex; D015870:Gene Expression / Q000502:physiology; D006056:Golgi Apparatus / Q000737:chemistry* / Q000378:metabolism; D007614:Macropodidae; D008868:Microtubule Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D011108:Polymers; D011233:Precipitin Tests; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry; D014162:Transfection
D000199:Actins; D015415:Biomarkers; D000072159:Dynactin Complex; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D011108:Polymers; D013049:Spectrin; C076834:centractin