Nuclear Import and Export Signals of Human Cohesins SA1/STAG1 and SA2/STAG2 Expressed in Saccharomyces cerevisiae

Abstract Background: Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown. Methodology/Principal Findings: In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the Nterminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus. Conclusions/Significance: This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells

A compound C-terminal nuclear localization signal of human SA2 stromalin

Stromalins are evolutionarily conserved multifunctional proteins with the best known function in sister chromatid cohesion. Human SA2 stromalin, likely involved in the establishment of cohesion, contains numerous potential nuclear localization (NLS) and nuclear export signals (NES). Previously we have found that the C-terminus of SA2 contains NLS(s) functional in human cells. However, the identity of this signal remained unclear since three NLS-like sequences are present in that region. Here we analyzed the functionality of these putative signals by expressing GFP-tagged C-terminal part of SA2 or its fragments in a human cell line and in the yeast Saccharomyces cerevisiae. We found that in human cells the nuclear import is dependent on a unique compound di- or tripartite signal containing unusually long linkers between clusters of basic amino acids. Upon expression of the same SA2 fragment in yeast this signal is also functional and can be easily studied in more detail

A selective autophagy cargo receptor NBR1 modulates abscisic acid signalling in Arabidopsis thaliana

The plant selective autophagy cargo receptor neighbour of breast cancer 1 gene (NBR1) has been scarcely studied in the context of abiotic stress. We wanted to expand this knowledge by using Arabidopsis thaliana lines with constitutive ectopic overexpression of the AtNBR1 gene (OX lines) and the AtNBR1 Knock-Out (KO lines). Transcriptomic analysis of the shoots and roots of one representative OX line indicated differences in gene expression relative to the parental (WT) line. In shoots, many differentially expressed genes, either up- or down-regulated, were involved in responses to stimuli and stress. In roots the most significant difference was observed in a set of downregulated genes that is mainly related to translation and formation of ribonucleoprotein complexes. The link between AtNBR1 overexpression and abscisic acid (ABA) signalling was suggested by an interaction network analysis of these differentially expressed genes. Most hubs of this network were associated with ABA signalling. Although transcriptomic analysis suggested enhancement of ABA responses, ABA levels were unchanged in the OX shoots. Moreover, some of the phenotypes of the OX (delayed germination, increased number of closed stomata) and the KO lines (increased number of lateral root initiation sites) indicate that AtNBR1 is essential for fine-tuning of the ABA signalling pathway. The interaction of AtNBR1 with three regulatory proteins of ABA pathway (ABI3, ABI4 and ABI5) was observed in planta. It suggests that AtNBR1 might play role in maintaining the balance of ABA signalling by controlling their level and/or activity

NLS of H2B does not confer nuclear localization on SA2S.

<p>(A) Yeast cells expressing fusion protein H2B^1–62-SA2S-GFP. (B) H2B^1–62-SA2S-GFP protein has predicted molecular weight. Diploid yeast strain <i>irr1Δ</i>/<i>IRR1</i> (lacking one copy of <i>IRR1</i> gene) was transformed with centromeric plasmid pUG35 bearing hybrid gene encoding the fusion protein. Details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038740#pone-0038740-g001" target="_blank">Figure 1</a>.</p

SA2S shuttles between nucleus and cytoplasm in yeast cells.

<p>(A) Subcellular localization of SA2S-GFP was analyzed after addition of LMB (Crm1p inhibitor) to 40 ng/ml to cells in logarithmic phase of growth. Strain <i>CRM1-T539C</i> bears LMB-sensitive version of Crm1p. Fourth column shows a composite of two fields from a single experiment but photographed as separate images, as marked. (B) Localization of SA2S-GFP protein was analyzed in thermo-sensitive <i>crm1-1</i> mutant. Transfer of cells grown at 30°C to 37°C for 30 minutes caused nuclear shift of the fusion protein in 100% of cells. Third and fourth columns show a composite of two fields from a single experiment, as marked. On the right in (A) and (B) control experiments in wild-type yeast are shown. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. (C) Frequencies of cells localized predominantly to the cytoplasm (black) or to the nucleus (gray) in strains bearing <i>CRM1-T539C</i> (LMB-sensitive) or <i>crm1-1</i> (thermo-sensitive) versions of Crm1p, following LMB treatment or temperature shift, respectively. MN47 and ABL10 are corresponding control strains bearing wild type <i>CRM1</i> gene, subjected to the same treatments.</p

SA1 contains NLS functional in yeast between 34K and 53K.

<p>(A) – Cells expressing SA1Δ34–53-GFP. (B) – Cells expressing fusion protein SA1^1–71-GFP. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. Column (A) shows a composite of two fields from a single experiment but photographed as separate images, as marked. For subcellular localization of intact.</p

Plasmids used in this study.

<p>Abbreviations for description of plasmids: CEN, centromeric; 2 µ, episomal; MCS, multiple cloning site.</p

SA2S contains NES functional in yeast between L953 and M962.

<p>Consensus for Crm1p-dependent export: ΦX_2–3ΦX_2–3ΦXΦ, where Φ represents L, I, V, F or M and X – any amino acid. (A) Left – cells expressing fusion protein SA2S-GFP. Right – cells expressing SA2SF960E–GFP protein bearing the substitution F960E which disrupts NES 953 LEK FMT <u>F</u>QM 962. The SA2SF960E–GFP protein accumulates in the nucleus in 100% of cells. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. (B) SA2S-GFP protein with NES signals disrupted by site-directed mutagenesis. SA2V699S – inactivated NES between positions 689 and 699, SA2SF960E – between positions 953–964. At least 100 cells were counted. Bars show percentage of cells with a given localization of GFP signal. Protein localized to the cytoplasm – black, to the nucleus–grey.</p

NLS of SA1 identified in yeast is the same in HeLa cells but SA2 is targeted to the nucleus of HeLa by C-terminally localized NLS.

<p>(A) Schematic representation of HeLa cells expressing SA1-GFP and SA2-GFP and their deletion mutants. Arrows indicate localization of NLS discussed in the text. Grey color indicates GFP fluorescence. (B) HeLa cells expressing SA1-, SA2L- and SA2S-GFP fusion proteins. (C) HeLa cells expressing SA1-GFP and SA2L-GFP devoid of N-terminal NLS 34–53 and 32–47, respectively. (D) HeLa cells expressing SA2L protein devoid of 161 C-terminal amino acids (upper panel), C-terminal NLS 1071–1140 (middle panel), C-terminal NLS 1199–1206 (lower panel). GFP represents fluorescence of fusion proteins, DNA was stained with DAPI.</p