C-TERMINAL TRUNCATIONS OF THE YEAST NUCLEOPORIN NUP145P PRODUCE A RAPID TEMPERATURE-CONDITIONAL MESSENGER-RNA EXPORT DEFECT AND ALTERATIONSTO NUCLEAR-STRUCTURE

A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)(+) RNA has iden tified an allele of the NUP145 gene, which encodes an essential nucleo porin. NUP145 was previously identified by using a genetic synthetic l ethal screen (E. Fabre, W. C. Boelens, C. Wimmer, I. W. Mattaj, and E. C. Hurt, Cell 78:275-289, 1994) and by using a monoclonal antibody wh ich recognizes the GLFG family of vertebrate and yeast nucleoporins (S . R. Wente and G. Blobel, J. Cell Biol, 125:955-969, 1994), Cells carr ying the new allele, nup145-10, grew at 23 and 30 degrees C but were u nable to grow at 37 degrees C, Many cells displayed a modest accumulat ion of poly(A)(+) RNA under permissive growth conditions, and all cell s showed dramatic and rapid nuclear accumulation of poly(A)(+) RNA fol lowing a shift to 37 degrees C. The mutant allele contains a nonsense codon which truncates the 1,317-amino-acid protein to 698 amino acids, This prompted us to examine the role of the carboxyl half of Nup145p. Several additional alleles that encode C-terminally truncated protein s or proteins containing internal deletions of portions of the carboxy l half of Nup145p were constructed, Analysis of these mutants indicate s that some sequences between amino acids 698 and 1095 are essential f or RNA export and for growth at 37 degrees C, In these strains, nuclea r accumulation of poly(A)+ RNA and fragmentation of the nucleolus occu rred rapidly following a shift to 37 degrees C. Constitutive defects i n nuclear pore complex distribution and nuclear structure were also se en in these strains, Although cells lacking Nup145p grew extremely slo wly at 23 degrees C and did not grow at 30 degrees C, efficient growth at 23 or 30 degrees C occurred as long as cells produced either the a mino 58% or the carboxyl 53% of Nup145p. Strains carrying alleles of N UP145 lacking up to 200 amino acids from the carboxy terminus were via ble at 37 degrees C but displayed nucleolar fragmentation and some nuc lear accumulation of poly(A)(+) RNA following a shift to 37 degrees C. Surprisingly, these strains grew efficiently at 37 degrees C in spite of a reduction in the level of synthesis of rRNAs to approximately 25 % of the wild-type level.