MOLECULAR AND FUNCTIONAL-CHARACTERIZATION OF THE P62 COMPLEX, AN ASSEMBLY OF NUCLEAR-PORE COMPLEX GLYCOPROTEINS

Macromolecular trafficking across the nuclear envelope involves intera ctions between cytosolic transport factors and nuclear pore complex pr oteins, The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carr ied out cDNA cloning of rat p58, p54, and p45. We found that p58 conta ins regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its N H2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a C OOH-terminal predicted coiled-coil region. p45 and p58 appear to be ge nerated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold e lectron microscopy, we found that p58/p45 and p54 are localized on bot h sides of the nuclear pore complex, like p62. Previous studies have s hown that immobilized recombinant p62 can bind the cytosolic nuclear i mport factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can d eplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF 2, At least in the case of p58, this involves FG repeat regions. Moreo ver, p58 can bind to a complex containing transport ligand, the nuclea r localization sequence receptor (Srp1 alpha) and p97, These data supp ort a model in which the p62 complex binds to a multicomponent particl e consisting of transport ligand and cytosolic factors to achieve accu mulation of ligand near the central gated channel of the nuclear pore complex.