Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles.

Abstract

Abstract. Centractin (Arpl), 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 ([3IE*) 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 resuits 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. CENTRACTIN is an actin-related protein (Arp) 1 that has been defined as a member of the Arpl class (Frankel et al., 1994;Fyrberg et al., 1994;Schroer et al., 1994). Actin-related proteins share 30-62% sequence identity with conventional actin and are predicted to have similar core structures for nucleotide binding (Kabsch and Holmes, 1995;Kelleher et al., 1995). The actin-related proteins have been grouped into three classes , however, the extent of the actinrelated protein family is not yet clear. To date, over 20 actin-related proteins have been identified in eukaryotes; an actin-related protein in protists has also been reported (Guerrero et al., 1995). The actin-related proteins appear to have important, but diverse cellular functions. Arpl null mutants in S. cerevisiae have defects in nuclear migration and mitotic spindle orientation

Evidence for a Functional Interaction between the ClC-2 Chloride Channel and the Retrograde Motor Dynein Complex

“…On the other hand, it is well known that dynactin interacts with and activates cytoplasmic dynein (33). Further, it has been shown that dynactin may recruit dynein to cargo in membrane vesicles directly (34) or indirectly via cytoskeletal proteins such as spectrin (35)(36)(37). Hence, in our future work, we will test the prediction that dynactin and spectrin may mediate the interaction between ClC-2 and dynein.…”

The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p

“…The only other known adapter that links a motor to its cargo is dynactin, a 20S multisubunit complex that interacts with cytoplasmic dynein (16)(17)(18). Dynactin is important for docking dynein onto various types of cargo, including the Golgi apparatus (19), lysosomes (20), and kinetochores (21). Similarly, it will be interesting to establish whether She3p serves as a general Myo4p adapter for various cargo besides Ash1 mRNA.…”

βIII Spectrin Binds to the Arp1 Subunit of Dynactin

“…Dynactin might interact directly with components of the lipid bilayer (13), but extraction studies suggest that dynactin is more peripherally associated with organelle membranes via protein-protein interactions (1). Based on observations of cells overexpressing the Arp1 subunit of dynactin, we previously proposed that the association of dynactin with vesicles is mediated by an interaction between Arp1 and an organelle-associated spectrin (11,14).Conventional spectrin associates with the cytoplasmic face of the plasma membrane, forming orthogonal arrays that are connected by 37-nm actin filaments and that are attached to the membrane by peripheral and integral membrane proteins (15, 16). A noncortical role for spectrin has also been proposed.…”

“…Dynactin might interact directly with components of the lipid bilayer (13), but extraction studies suggest that dynactin is more peripherally associated with organelle membranes via protein-protein interactions (1). Based on observations of cells overexpressing the Arp1 subunit of dynactin, we previously proposed that the association of dynactin with vesicles is mediated by an interaction between Arp1 and an organelle-associated spectrin (11,14).…”

“…One possibility is that dynactin increases the processive nature of the movement of dynein along cellular microtubules (3,10). A second but not mutually exclusive hypothesis is that dynactin links dynein to its cellular cargo (11).…”