Translocation through the nuclear pore complex (NPC) is catalyzed by a remarkable gating mechanism that can dilate to accommodate a range of cargo sizes. We have correlated mutations of certain nonessential yeast nucleoporin genes with large increases in NPC permeability, thereby implicating specific nucleoporins (nups) in channel gating. We then found that the increased permeability in nup170-delta cells, for example, is associated with the complete but reversible dissociation Nup188p, Nup53p, Nup120p from the NPC. Therefore, increases in the diameter of the central channel are correlated with the disruption of specific nup-nup complexes. This gate-like mechanism is likely to be conserved because NUP170, NUP188, and other components of the putative yeast gating apparatus are conserved in vertebrates. Experiments aimed at generalizing and elucidating the molecular mechanism of NPC channel gating will use yeast molecular genetics, in vitro studies with purified proteins, and permeabilized mammalian tissue culture cells. A second line of research implicates Hsp70 in the closing or rectification of the central channel. Increases in NPC permeability and the reversible dissociation of nups in mutant cells are suppressed by inducing Hsp70. These and other results suggest that Hsp70 facilitates channel closing by catalyzing reversible conformational changes that switch the NPC between open and closed states. An unexpected prediction of this hypothesis is that channel closing requires ATP hydrolysis by Hsp70. By analogy, ER lumenal Hsp70 (BIP) catalyzes closure of the secretion translocon during integration of membrane spanning domains. The long-term goal of this proposal is to elucidate the molecular mechanism of NPC channel gating. Specifically, we propose to define at a biochemical level the specific nup-nup interactions that are reversibly broken during gating. Also, we propose to test the hypothesis that Hsp70 catalyzes the reforming of the specific nup-nup bonds that characterize the channel s closed conformation(s). Nuclear transport is a fundamental cell process that is intimately involved in gene expression and is relevant to issues of human disease and health.
