Yeast karyopherin Kap95 is required for cell cycle progression at Start

Abstract

Background: The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in cell division regulation. The active transport of proteins between the nucleus and the cytoplasm is mediated by the transport receptors of the beta-karyopherin family. In this work we characterized the terminal phenotype of a mutant strain in beta-karyopherin Kap95, a component of the classical nuclear import pathway.

Results: When KAP95 was inactivated, most cells arrested at the G2/M phase of the cell cycle, which is in agreement with the results observed in mutants in the other components of this pathway. However, a number of cells accumulate at G1, suggesting a novel role of Kap95 and the classical import pathway at Start. We investigated the localization of Start transcription factors. It is known that Swi6 contains a classical NLS that interacts with importin alpha. Here we show that the in vivo nuclear import of Swi6 depends on Kap95. For Swi4, we identified a functional NLS between amino acids 371 and 376 that is sufficient and necessary for Swi4 to enter the nucleus. The nuclear import driven by this NLS is mediated by karyopherins Kap95 and Srp1. Inactivation of Kap95 also produces a dramatic change in the localization of Mbp1 since the protein is mainly detected in the cytoplasm. Two functionally redundant Kap95- and Srp1-dependent NLSs were identified in Mbp1 between amino acids 27-30 and 166-181. Nuclear accumulation was not completely abolished in a kap95 mutant or in the Mbp1 mutated in the two NLSs, suggesting that alternative pathways might contribute to the Mbp1 nuclear import to a lesser extent.

Conclusions: Kap95 plays an essential role at the initiation of the cell cycle by driving the nuclear import of Swi4, Swi6 and Mbp1, the three transcription factors responsible for the gene expression at Start. This transport depends on the specific nuclear localization signals present in cargo proteins.

Figure 1

Characterization of the terminal phenotype of tetO ₇:KAP95 mutant strain. Exponentially growing cells of the wild type (W303-1a) and tetO ₇:KAP95 (JCY635) strains were incubated in the presence of 5 μg/mL doxycycline for 6 hours. (A) DIC and DAPI staining of DNA images. Graph shows distribution of cells based on the bud size. (B) FACS analysis of DNA content. (C) Distribution of large budded cells based on number and location of nuclei. (D) Exponentially growing cells of the cdc15 and cdc15 tetO ₇:KAP95 (JCY1543) strains were incubated in the presence of 5 μg/mL doxycycline at 37° for 4 hours. After transfer to 25°, cell morphology and nuclei number were analyzed at the indicated times (see text). (E) Cell morphology and nuclei number were analyzed in the samples of the cdc15 tetO ₇:KAP95 (JCY1543) strain at 0 and 120 min after digestion of cell wall with zymolyase.

Figure 2

Analysis of Start transcription in the kap95 mutant strain. (A) Northern analysis of the expression of the Start genes CLN2 and RNR1 in exponentially growing cells of the wild type (W303-1a) and tetO ₇:KAP95 (JCY635) strains incubated in the presence of 5 μg/mL doxycycline for 6 hours. SRC1 transcript is shown as a loading control. (B) Exponentially growing cells of the cdc15 and cdc15 tetO ₇:KAP95 (JCY1543) strains were incubated in the presence of 5 μg/mL doxycycline at 37° for 4 hours. After transfer to 25°, expression of CLN2 and RNR1 genes at the indicated times was analyzed by northern analysis (C) Exponentially growing cells of the tetO ₇:KAP95 (JCY635) strain transformed with a control plasmid (pRS313) or a plasmid expressing the CLN2 gene under the control of the S. pombe adh1 promoter were incubated in the presence of 5 μg/mL doxycycline for 6 hours. The graph shows the distribution of cells based on the bud size. (D) Exponentially growing cells of the cdc15 tetO ₇:KAP95 (JCY1543) strain transformed with a control (pRS313) or the adh1:CLN2 plasmid were incubated in the presence of 5 μg/mL doxycycline at 37° for 4 hours. After transfer to 25°, cell morphology was analyzed at the indicated times.

Figure 3

Subcellular localization of Swi6 in the kap95 mutant strain. Cells from exponentially growing cultures of the wild type (JCY710) and tetO ₇:KAP95 (JCY637) strains expressing the HA-tagged version of Swi6, were incubated in the presence of 5 μg/mL doxycycline for 6 hours and assayed by indirect immunofluorescence. The HA indirect-fluorescence signals, the DAPI staining of DNA and DIC images are shown. No signal was detected in a control of the untagged W303-1a strain.

Figure 4

Control of the subcellular localization of Swi4. (A) A putative NLS sequence in the Swi4 protein is indicated (white box). Amino acids substitutions introduced to inactivate the NLS are shown in grey. Grey boxes represent the DNA binding (DBD), the ankyrin (ANK) and the Swi6 binding (SBD) domains. (B) Exponentially growing cells of the wild type strain (W303-1a) transformed with plasmids pSWI4^NLS-GFP₄ and pSWI4^NLSi-GFP₄ were analyzed by fluorescence microscopy. GFP signal images are shown. (C) Exponentially growing cells of the wild type (W303-1a) and tetO₇:KAP95 (JCY635) strains transformed with plasmid pSWI4^NLS-GFP₄ were incubated in the presence of 5 μg/mL doxycycline for 6 hours and analyzed by fluorescence microscopy. GFP signal images are shown. (D) Exponentially growing cells of the wild type strain (W303-1a) transformed with plasmids pGAL1:HA-SWI4 and pGAL1:HA-SWI4^NLSi were incubated in the presence of 5 μg/mL doxycycline for 6 hours and assayed by indirect immunofluorescence. The HA indirect-fluorescence signals, the DAPI staining of DNA and DIC images are shown. (E) Exponentially growing cells of the the wild type (W303-1a) and tetO ₇:KAP95 (JCY635) strains transformed with plasmid pGAL1:HA-SWI4 were incubated in the presence of 5 μg/mL doxycycline for 6 hours and assayed by indirect immunofluorescence. The HA indirect-fluorescence signals, the DAPI staining of DNA and DIC images are shown. (F) Exponentially growing cells of the the wild type (W303-1a) and the srp1 strains transformed with the plasmid pGAL1:HA-SWI4 were incubated at 37° for 2 hours and and assayed by indirect immunofluorescence. The wild type cells showed a nuclear localization of Swi4 similar to that showed in (E) and are not shown.

Figure 5

Control of the subcellular localization of Mbp1. (A) Cells from exponentially growing cultures of the wild type (JCY816) and tetO ₇:KAP95 (JCY848) strains expressing the HA-tagged version of Mbp1, were incubated in the presence of 5 μg/mL doxycycline for 6 hours and assayed by indirect immunofluorescence. The HA indirect-fluorescence signals, the DAPI staining of DNA and DIC images are shown. (B) Putative NLSs sequences in the Mbp1 protein are indicated as white boxes. Amino acids substitutions introduced to inactivate the NLSs are shown in grey. Grey boxes represent the DNA binding (DBD), the ankyrin (ANK) and the Swi6 binding (SBD) domains. (C) Exponentially growing cells of the wild type strain (W303-1a) transformed with plasmids pMBP1^23-184-GFP4, pMBP1^23-184NLS1i-GFP4, pMBP1^23-184NLS2i-GFP4 and pMBP1^{23-184NLS1i2i}-GFP4 were analyzed by fluorescence microscopy. GFP signal images are shown. (D) Exponentially growing cells of of the wild type (W303-1a) and tetO₇:KAP95 (JCY635) strains transformed with plasmid pMBP1^23-184-GFP4 were incubated in the presence of 5 μg/mL doxycycline for 6 hours and analyzed by fluorescence microscopy. GFP signal and DIC images are shown. (E) Exponentially growing cells of the the wild type (W303-1a) and the srp1 strains transformed with plasmid pMBP1^23-184-GFP4 were incubated at 37° for 2 hours. GFP signal images are shown.

Figure 6

Coordinated spatial regulation of Start transcriptional machinery. Expression of genes at the G1/S transition is regulated by the transcription factors SBF (Swi4-Swi6), MBF (Mbp1-Swi6) and the transcriptional repressor Whi5. The four proteins Swi4, Swi6, Mbp1 and Whi5 are imported into the nucleus by the same pathway involving karyopherin Kap95 and classical NLS sequences. Swi4 and Mbp1 remains nuclear along the cell cycle. However, after Start activation Whi5 and Swi6 are exported to the cytoplasm by karyopherin Msn5, a transport that depends on the phosphorylation of the proteins. At the end of mitosis, the Cdc14 phosphatase dephoshorylates Whi5 and Swi6 to once again lead to the nuclear acumulation of the proteins.