Dynein Light Chain Association Sequences Can Facilitate Nuclear Protein Import

Abstract

Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application. INTRODUCTIONNuclear protein transport is central to normal and aberrant cellular development and physiology, as well as the infectious cycles of intracellular pathogens Hearps and Jans, 2006). The ability to exploit the cellular factors involved in nuclear targeting is of great therapeutic value as it permits the design of vehicles for the efficient delivery of drugs/therapeutic genes to the nuclear compartment (Chan and Jans, 2002; Mastrobattista et al., 2006a,b). All trafficking across the nuclear membrane occurs through the nuclear membrane embedded nuclear pore complex (NPC). The movement of molecules Ͼ45 kDa generally requires active transport, where proteins conventionally interact via a nuclear localization sequence (NLS) with import proteins (importins) which mediate docking to and transit through the NPC . Current understanding of this process focuses largely on these events at the NPC because investigation of nuclear import has conventionally used permeabilized cell systems with associated disruption of cytoplasmic structures. Thus, it is only through recent studies with live cells that it has become clear that nuclear import can involve additional levels of regulation in the cytoplasm involving the microtubule (MT) cytoskeleton (Giannakakou et al., 2000;Lam et al., 2002;Roth et al., 2007), a network composed of ϩ/Ϫ polarized polymers of tubulin with the negative end at the MT organizing center (MTOC) located near the nucleus of nonpolarized cells. MTs are suggested to act as tracks for cargo delivery by molecular motors including the multiprotein complex motor, dynein, which mediates net movement of cargoes toward the MTOC (retrograde movement),...

Stimulated nuclear translocation of NF-κB and shuttling differentially depend on dynein and the dynactin complex

“…Consensus sequences conferring interaction with the dynein light chains LC8 and Tctex-1 are beginning to be defined. Our analysis of human and mouse p65 protein indicate that no known sites appear present; however, the interaction sites of most dynein cargo remain uncharacterized (29,31).…”

“…Interestingly, dynein knockdown did not significantly alter either CREB or NFAT reporter activity, although it has been reported that perturbations in the microtubule network could inhibit NFAT (32). While p53 is known to use dynein (26), the transport mechanisms of many nuclear localizing molecules are either unknown or are independent of the cytoskeleton (29,33,34). The further understanding of both dynein-dependent and independent mechanisms of NF-B transport may reveal additional regulatory points in this widespread signaling pathway.…”

“…While microtubule-dependent transport of vesicles and other large cargo has been well studied, several reports have highlighted microtubule-dependent transport of small nonvesicular cargo and have also linked dynactin and dynein to nonvesicular protein transport (26)(27)(28)(29). A microinjected complex of the p50 NF-B subunit with plasmid DNA was reported to enhance nuclear import and cytoplasmic migration of the DNA with a dependence on microtubules, dynein, and the p50 NLS (30); sequences distinct from a NLS bind dynein, and the presence of an NLS can, but does not necessarily, confer microtubuledependent transport (29).…”

“…A microinjected complex of the p50 NF-B subunit with plasmid DNA was reported to enhance nuclear import and cytoplasmic migration of the DNA with a dependence on microtubules, dynein, and the p50 NLS (30); sequences distinct from a NLS bind dynein, and the presence of an NLS can, but does not necessarily, confer microtubuledependent transport (29). Consensus sequences conferring interaction with the dynein light chains LC8 and Tctex-1 are beginning to be defined.…”

Transport and egress of herpes simplex virus in neurons

“…In fact, it is hard to envisage what role any of these interactions with dynein would play in the replication of HSV-1. One explanation may be that dynein plays a role in the nuclear import of pUL34 and pUL9, as observed for other VPs [61].…”