A structure/function analysis of Rat7p/Nup159p, an essential nucleoporin of Saccharomyces cerevisiae.
Rat7p/Nup159p is an essential nucleoporin of Sac-charomyces cerevisiae originally isolated in a genetic screen designed to identify yeast temperature-sensitive mutants defective in mRNA export. Here we describe a detailed structural-functional analysis of Rat7p/Nup159p. The mutation in the rat7-1 ts allele, isolated in the original genetic screen, was found to be a single base pair change that created a stop codon approximately 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 completely dispensable for growth at temperatures between 16 degrees C and 37 degrees C and cells expressing this mutant allele were indistinguishable from wild type. Deletion of the amino-terminal third of the protein, upstream from the repeat region, or the portion between the repeat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the behavior of nuclear pore complexes (NPCs). Taken together, our data suggest that Rat7p/Nup159p is anchored within the NPC through its coiled-coil region and adjacent sequences. In addition, we postulate that the N-terminal third of Rat7p/Nup159p plays an important role in mRNA export.
Del Priore
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Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Heath
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Snay
C
C
MacMillan
A
A
Gorsch
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Dagher
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S
Cole
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C
D000483:Alleles; D019943:Amino Acid Substitution; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry; D005800:Genes, Fungal; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012331:RNA, Fungal / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion
D005656:Fungal Proteins; D008565:Membrane Proteins; C093510:NUP159 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins