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J. Cell Biol.,
Volume 144, Number 6, March 22, 1999 1097-1112
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021
The mammalian nuclear pore complex
(NPC) is comprised of ~50 unique proteins, collectively
known as nucleoporins. Through fractionation of rat
liver nuclei, we have isolated >30 potentially novel nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel nucleoporin with a predicted
molecular mass of 96 kD. Nup96 is generated through
an unusual biogenesis pathway that involves synthesis
of a 186-kD precursor protein. Proteolytic cleavage of
the precursor yields two nucleoporins: Nup98, a previously characterized GLFG-repeat containing nucleoporin, and Nup96. Mutational and functional analyses
demonstrate that both the Nup98-Nup96 precursor and
the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are
proteolytically cleaved in vivo. This biogenesis pathway
for Nup98 and Nup96 is evolutionarily conserved, as
the putative Saccharomyces cerevisiae homologues,
N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the
nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96
and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex
containing Nup96, Nup107, and at least two Sec13-
related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast
and mammals.
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