ABSTRACT
The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In Saccharomyces cerevisiae, Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus.
Subject(s)
Exosome Multienzyme Ribonuclease Complex/metabolism , Exosomes/metabolism , Karyopherins/metabolism , Nuclear Localization Signals/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , beta Karyopherins/metabolism , Active Transport, Cell Nucleus , Exosome Multienzyme Ribonuclease Complex/chemistry , Exosome Multienzyme Ribonuclease Complex/genetics , Exosomes/enzymology , Gene Deletion , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Karyopherins/chemistry , Karyopherins/genetics , Microscopy, Confocal , Microscopy, Fluorescence , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , beta Karyopherins/chemistry , beta Karyopherins/geneticsABSTRACT
Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi using real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point.