A STRUCTURE FUNCTION ANALYSIS OF RAT7P/NUP159P, AN ESSENTIAL NUCLEOPORIN OF SACCHAROMYCES-CEREVISIAE/

Rat7p/Nup159p is an essential nucleoporin of Saccharomyces cerevisiae originally isolated in a genetic screen designed to identify yeast tem perature-sensitive mutants defective in mRNA export, Here we describe a detailed structural-functional analysis of Rat7p/Nup159p. The mutati on in the rat7-1 ts allele, isolated in the original genetic screen, w as found to be a single base pair change that created a stop codon app roximately 100 amino acids upstream of the actual stop codon of this 1 ,460 amino acid polypeptide, thus eliminating one of the two predicted coiled-coil regions located near the carboxyl terminus of the protein , These coiled-coil regions are essential since an allele lacking both coiled-coil regions was unable to support growth under any conditions , In contrast, no other region of the protein was absolutely required, The SAFG/PSFG repeat region in the central third of the protein was c ompletely dispensable for growth at temperatures between 16 degrees C and 37 degrees C and cells expressing this mutant allele were indistin guishable from wild type, Deletion of the aminoterminal third of the p rotein, upstream from the repeat region, or the portion between the re peat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the beh avior of nuclear pore complexes (NPCs), Taken together, our data sugge st that Rat7p/Nup159p is anchored within the NPC through its coiled-co il region and adjacent sequences, In addition, we postulate that the N -terminal third of Rat7p/Nup159p plays an important role in mRNA expor t.