Yeast Krr1p physically and functionally interacts with a novel essential Kri1p, and both proteins are required for 40S ribosome biogenesis in the nucleolus

Mol Cell Biol. 2000 Nov;20(21):7971-9. doi: 10.1128/MCB.20.21.7971-7979.2000.

Abstract

Using a two-hybrid screening with TOM1, a putative ubiquitin-ligase gene of Saccharomyces cerevisiae, we isolated KRR1, a homologue of human HRB2 (for human immunodeficiency virus type 1 Rev-binding protein 2). To characterize the gene function, we constructed temperature-sensitive krr1 mutants and isolated two multicopy suppressors. One suppressor is RPS14A, encoding a 40S ribosomal protein. The C-terminal-truncated rpS14p, which was reported to have diminished binding activity to 18S rRNA, failed to suppress the krr1 mutant. The other suppressor is a novel gene, KRI1 (for KRR1 interacting protein; YNL308c). KRI1 is essential for viability, and Kri1p is localized to the nucleolus. We constructed a galactose-dependent kri1 strain by placing KRI1 under control of the GAL1 promoter, so that expression of KRI1 was shut off when transferring the culture to glucose medium. Polysome and 40S ribosome fractions were severely decreased in the krr1 mutant and Kri1p-depleted cells. Pulse-chase analysis of newly synthesized rRNAs demonstrated that 18S rRNA is not produced in either mutant. However, wild-type levels of 25S rRNA are made in either mutant. Northern analysis revealed that the steady-state levels of 18S rRNA and 20S pre-rRNAs were reduced in both mutants. Precursors for 18S rRNA were detected but probably very unstable in both mutants. A myc-tagged Kri1p coimmunoprecipitated with a hemagglutinin-tagged Krr1p. Furthermore, the krr1 mutant protein was defective in its interaction with Kri1p. These data lead us to conclude that Krr1p physically and functionally interacts with Kri1p to form a complex which is required for 40S ribosome biogenesis in the nucleolus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Northern
  • Cell Nucleolus / metabolism*
  • Cycloheximide / pharmacology
  • Fluorescent Antibody Technique, Indirect
  • Fungal Proteins / metabolism*
  • Galactose / metabolism
  • Gene Deletion
  • Homeodomain Proteins / metabolism
  • Immunoglobulin G / metabolism
  • Magnesium / metabolism
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism*
  • Plasmids / metabolism
  • Polyribosomes / metabolism
  • Precipitin Tests
  • Promoter Regions, Genetic
  • Protein Binding
  • RNA, Ribosomal / metabolism
  • RNA, Ribosomal, 18S / metabolism
  • RNA-Binding Proteins*
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism*
  • Ribosomes / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins*
  • Suppression, Genetic
  • Temperature
  • Two-Hybrid System Techniques
  • Ubiquitin-Protein Ligases

Substances

  • Fungal Proteins
  • Homeodomain Proteins
  • Immunoglobulin G
  • KRR1 protein, S cerevisiae
  • Nuclear Proteins
  • RNA, Ribosomal
  • RNA, Ribosomal, 18S
  • RNA-Binding Proteins
  • Ribosomal Proteins
  • Saccharomyces cerevisiae Proteins
  • ribosomal protein S40
  • RNA, ribosomal, 25S
  • Cycloheximide
  • TOM1 protein, S cerevisiae
  • Ubiquitin-Protein Ligases
  • Magnesium
  • Galactose