Molecular and Functional Basis for the Scaffolding Role of the p50/Dynamitin Subunit of the Microtubule-associated Dynactin Complex

Abstract

p50/dynamitin (DM) is a major subunit of the microtubuleassociated dynactin complex that is required for stabilization and attachment of its two distinct structural domains, namely the Arp1 rod and the shoulder/sidearm. Here, we define the determinants of p50/DM required for self-oligomerization of the protein and for interactions with other subunits of the dynactin complex. Whereas the N-terminal 1-91-amino acid region of the protein is required and sufficient for binding to the Arp1 rod, additional determinants contained within the first half of the protein are required for optimal recruitment of the p150Glued subunit of the shoulder/sidearm. Overexpression experiments confirmed that the N-terminal 1-91-amino acid region of p50/DM is critical for dynactin functionality, because this fragment acts as a dominant negative to inhibit both dynein-dependent and -independent functions of the complex. Furthermore, the first two predicted coiled-coil motifs of p50/DM contain determinants that mediate selfassociation of the protein. Interestingly, p50/DM self-association does not contribute to p50/DM-induced disruption of the dynactin complex, but most likely participates in the stabilization of the complex.The dynactin complex was initially identified as a multiprotein complex co-purifying with the minus-end-directed microtubular motor dynein and capable of activating membrane vesicle movements (1, 2). Initial genetic studies performed in yeast, filamentous fungi, and Drosophila demonstrated that dynactin was an obligate cofactor of the dynein motor (3-6). To date, many functional studies confirmed that the dynactin complex is required for most, if not all, dynein-based cellular processes (reviewed in Ref. 7). During interphase, dynactin plays a major role in all dynein-based motile events by providing both stable cargo binding and processivity activities, to ensure proper vesicular transport between cellular compartments along the microtubules (8 -11). Moreover, the dynactin complex was also shown to interact with and increase the processivity of the kinesin-II plus-ended motor, suggesting an unexpected role in plusend-directed motile events (12)(13)(14). In addition, dynactin plays other roles independently of microtubule motors, by facilitating attachment of microtubules to several subcellular structures. Although initial studies demonstrated that dynactin contributed to microtubule anchoring at the centrosome during interphase (15, 16), recent results provided the first clues concerning its role as an initial receptor for cytoplasmic membrane vesicles at microtubule plus ends (17-20), although this model has been brought into question (21).Dynactin is a multiprotein complex of ϳ1.2 MDa, composed of 11 different polypeptides that are present as single or multiple copies and organized in two distinct structural domains (see Fig. 1) as follows: (i) the Arp1 rod, consisting of a short polymer of eight copies of Arp1 (actin-related protein-1), capped on its positive barbed end by the CapZ ␣/␤ heterodimer and on its nega...

Dynactin integrity depends upon direct binding of dynamitin to Arp1

“…A later report indicated that AA 100–403, a fragment that contains all these motifs, did not cause organelle or spindle derangement when overexpressed (Jacquot et al ., 2010). We, too, found that overexpression of TAP-tagged dynamitin AA 100–403 did not trigger release of p150 Glued or endogenous dynamitin from the Arp filament and further observed that AA 100–403 did not cosediment with Arp1 when cytosols were subjected to sucrose gradient centrifugation (Supplemental Figure S2), suggesting that this fragment cannot bind the dynamitin protomers that are incorporated into dynactin.…”

“…However, fragments lacking some or all of these motifs are sufficient for disruption (Maier et al ., 2008; Jacquot et al ., 2010), indicating that dynamitin may be able to trigger dynactin disassembly via more than one mechanism. In the present study, we used direct protein–protein binding assays to characterize interactions between dynamitin's N-portion and the self-associating C-terminal portion with other dynactin subunits.…”

Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5

“…Interactions with partner proteins and oligomerization sites reported up to now are situated in the NtD2 [38,39]. The Cterminus of the protein seems to be indispensable for correct functioning of dynamitin, even though the role of this part of dynamitin remains obscure [38].…”

Rotational motion during three-dimensional morphogenesis of mammary epithelial acini relates to laminin matrix assembly

“…To genetically interfere with microtubule function we ovexpressed dynamitin and disrupted dynein function (Fig. S2A) (14,15). We observed a 75% decrease in the number of structures that rotated and a 50% decrease in the speed of movement (Fig.…”