A Novel Nuclear Pore Protein Nup82p Which Specifically Binds to a Fraction of Nsplp

Nsplp interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsplp, but does not co-purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsplp which is distinct from Nsplp found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsplp. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A) + RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsplp participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures. T HE major structural features of the nuclear pore complex have now been firmly established. Each nuclear pore complex (NPC) 1 is composed of the spoke complex sandwiched by an outer (cytoplasmic) and inner (nucleoplasmic) ring and the central plug (transporter). Attached to the inner ring is the nuclear basket and protruding from the outer ring are eight short filaments and particles (for review see Fabre and Hurt, 1994; Pant6 and Aebi, 1994; Rout and Wente, 1994). Some of these structures must be directly involved in bi-directional nucleocytoplasmic transport, which occurs exclusively through the nuclear pore complexes (Feldherr et al., 1984; Richardson et al., 1988). Concerning active nuclear import of proteins, which is NLS mediated and energy dependent, it was suggested that the central plug could serve as a transporter (Akey, 1992), whereas the channels between the spokes (Hinshaw et al., 1992) or between the transporter and the spokes (Akey and Radermacher, 1993) may allow passive diffusion of small molecules; furthermore, the short cytoplasmic filaments and the nuclear baskets were proposed to be involved in the early docking steps at The sequence data for the NUP82 gene are available from Genbank/ EMBL/DDBJ under accession number X85970. Address all correspondence to E. Hurt, EMBL, Meyerhofstrasse 1, Postfach 10 22 09, 69117 Heidelberg, Germany. Tel.: (49) 6221 387 363, Fax: (49) 6221. E. C. Hurt 's present address is University of Heidelberg, Institut fur Biochemie I, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. 1. Abbreviat ions used in this paper: NPC, nuclear pore complex; nt, nucleotide; TFA, triflnoroacetic acid; WGA, wheat germ agglutinin. the NPC for protein import and RNA export, respectively (Feldherr et al., 1984; Richardson et al., 1988; Mehlin et al., 1992). Whereas a mechanistic picture for the cytoplasmic phase of nuclear protein import is beginning to emerge (Powers and Forbes, 1994), very little is known about the following steps at the NPC, in particular which nuclear pore proteins are involved in the actual translocation process and how they act. It is likely that some of the cytosolic/nuclear factors required for nucleocytoplasmic transport such as the NLS receptor complex initially identified by Adam and Gerace (1991) and later shown to be importin 60/90 (G6rlich et al., 1994; G6rlich et al., 1995) which is also called karyopherin a/J3 (Moroianu et al., 1995; Radu et al., 1995), Hse70 (for review, see Goldfarb, 1992), the small GTPase Ran/TC4 (Moore and Blobel, 1994b), its guanine nucleotide exchanger RCC1 (Kadowaki et al., 1993; Tachibana et al., 1994), and the Ran-associated protein B2 (Moore and Blobel, 1994a) may transiently interact with NPC components during transport of the substrate through the NPC channel, but how this occurs is still unknown. The observation that substrates for nuclear import can bind to the nuclear envelope under conditions in which translocation is inhibited was taken as evidence that NLS receptor(s) can dock at the NPC presumably by binding to specific NPC proteins. Nuclear protein import is inhibited by microinjection of the lectin wheat germ agglutinin (WGA) that binds to a family of NPC proteins (Finlay et al., 1987; Dabauvalle et al., 1990). The interaction of this family of nuclear pore proteins with cytosolic transport factors is also © The Rockefeller University Press, 0021-9525/95/09/1263/11 $2.00 The Journal of Cell Biology, Volume 130, Number 6, September 1995 1263-1273 1263 on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published September 15, 1995