QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 20 November 2007
doi: 10.1242/jcs.019943

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Spiller, M. P. Articles by Beggs, J. D. Search for Related Content PubMed PubMed Citation Articles by Spiller, M. P. Articles by Beggs, J. D.

Requirements for nuclear localization of the Lsm2-8p complex and competition between nuclear and cytoplasmic Lsm complexes

Michael P. Spiller^*,, Martin A. M. Reijns^* and Jean D. Beggs

Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK

View larger version (41K): [in this window] [in a new window]   Fig. 1. Localization of Lsm1p, Lsm7p and Lsm8p. (A) Western blot showing 13myc-tagged Lsm1p, Lsm7p and Lsm8p from yeast strains MPS1, MPS2 and MPS3, respectively. (B) Localization of Lsm1-13myc, Lsm7-13myc and Lsm8-13myc in strains MPS1, MPS2 and MPS3, respectively. Fixed cells were stained with anti-Myc antibodies followed by Cy3-conjugated secondary antibody (red in merge). Nuclei were stained with DAPI (green in merge). For clarity, single channel images of DAPI and of immunofluorescence are shown in greyscale. Arrows indicate `holes' in the immunofluorescence at the positions of nuclei. Bars, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Localization of the Lsm proteins in the nup49-313 nuclear-import mutant and the xpo1-1 export mutant. Cultures were grown at 23°C to log phase and then at 37°C for a further 4.5 hours. In each case, a representative cell is shown. (A) Localization of Lsm7-13myc in MPS2 (wild type, WT) and MPS12 (nup49-313) at 23°C and 37°C. (B) Localization of Lsm8-13myc in MPS3 (WT) and MPS13 (nup49-313) at 23°C and 37°C. (C) Fluorescent in situ hybridization showed that localization of U1 snRNA in nup49-313 yeast at restrictive temperature was the same as in wild-type cells. (D) Localizations of Lsm1-13myc, Lsm7-13myc and Lsm8-13myc at 37°C in the xpo1-1 mutant strains MPS13, MPS14 and MPS15, respectively, were the same as for wild-type cells. Arrows indicate `holes' in the immunofluorescence at the positions of nuclei. Bars, 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Localization of Lsm7p upon depletion of other Lsm proteins. (A) Localization of Lsm7p in the absence of Lsm8p in the strain MPS7. MPS7 (PGAL1-HA-LSM8) cells were grown in galactose medium to mid-log-phase and then shifted to glucose for 12 hours to deplete Lsm8p (confirmed by western blotting; data not shown). Nuclei depleted of Lsm7p are indicated by a large arrow and cytoplasmic foci are indicated by small arrows. (B) Localization of Lsm7p in strains lacking other Lsm proteins. lsm1 and lsm6 strains (MPS6 and MPS4) were grown at permissive temperature in glucose, whereas MPS8, MPS9 and MPS10 were depleted of Lsm3p, Lsm4p and Lsm5p, respectively, by growth in glucose for 12 hours. Representative cells are shown. Bars, 10 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Localization of Lsm8p in the absence of Lsm2p, Lsm4p or Lsm6p. Localization of 13myc-tagged Lsm8p (red in merge) in wild-type (WT; MPS3) yeast, or after depleting Lsm2p or Lsm4p by growing MPS22 or MPS25 cells in glucose for 12 hours, or in strain MPS5 (which has the LSM6 gene knocked out) grown at permissive temperature. Bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Lsm8p nuclear localization is affected by C-terminal, N-terminal and Sm-site deletions. (A) Cartoon showing the structures of the Lsm8 deletion constructs that were tested as GFP-fusion proteins. (B) Localization of GFP-tagged Lsm8p constructs in yeast strain MPS26 plus (i) pMPS8 (GFP-Lsm8), (ii) pMPS8-82 (GFP–Lsm8-2), (iii) pMPS8-313 (GFP–Lsm8-313), (iv) pMPS8C (GFP-Lsm8Cterm), (v) pMPS8-6A3 (GFP–Lsm8-6A3), (vi) pMPS8-N (GFP-Lsm8N), (vii) pMPS8-SM (GFP-Lsm8Sm); or in (vii) BMA38a plus pMPS8 (i.e. GFP-Lsm8 produced in the presence of endogenous Lsm8p) or (viii) pGFP-N-FUS (GFP). In all cases, GFP localization was examined in live cells after growth in SD-Ura-Met (to induce expression of the GFP construct). Bar, 10 µm. (C) Co-immunoprecipitation of myc-tagged Lsm7p with GFP-Lsm8p. Lsm8p-containing complexes were precipitated with anti-GFP antibodies, and Lsm7p was detected with anti-myc antibodies. (D) All the GFP-Lsm8 constructs were stably expressed, as shown by western blotting (and data not shown) using anti-GFP antibodies.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Over-production of Lsm1p or Lsm8p has opposing effects on Lsm7p localization. (A) HA-tagged Lsm1p or Lsm8p was over-produced from plasmid pAEM80 or pAEM76, respectively, in strain MPS2 (Lsm7-13myc) grown in SDGal-Ura. Lsm7-13myc localization (red in merge) in wild type (WT; MPS2 carrying pBM125 empty vector), or with LSM1 or LSM8 overexpression (o/e LSM1 and o/e LSM8, respectively). Over-production of Lsm1p caused reduction of nuclear Lsm7p (large arrow) and its accumulation in foci (small arrows). Bar, 10 µm. (B) Lsm8p over-production does not affect Lsm1p levels. MPS1 was grown with pBM125 or pBM125-HA-LSM8 in SD-Ura (Glu) or SDGal-Ura (Gal). Total protein was separated by SDS-PAGE and the western blot was probed for Lsm1-13myc, HA-Lsm8 and -Tubulin.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Overexpression of LSM1 in an lsm8-1 background results in a severe slow-growth phenotype. (A) Growth curve of wild-type (WT; MPS2) and lsm8-1 (MPS17) strains with or without over-production of Lsm1p (1oe). Wild-type yeast with Lsm7p myc-tagged (MPS2) or lsm8-1 yeast with Lsm7p myc-tagged (MPS17) were transformed with either pBM125 (empty vector) or pAEM80 (PGAL1-HA-LSM1). All strains were grown in galactose-based media. (B) Northern blot showing the levels of U1 and U6 snRNAs in wild-type or lsm8-1 cells combined with over-production of Lsm1p. (C) Localization of Lsm7-13myc in wild-type and lsm8-1 strains, with or without over-production of Lsm1p. All strains were grown in galactose and the localization of Lsm7p was detected with anti-Myc antibodies. Representative cells are shown. Arrows indicate `holes' in the immunofluorescence at the positions of nuclei. Bars, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 8. Lsm7p and Lsm8p delocalize after hyperosmotic shock, but not after glucose deprivation. MRY74 (LSM7-13myc), MPS3 (LSM8-13myc), MPS15 (xpo1-1, LSM7-13myc) and MPS16 (xpo1-1 LSM8-13myc) cells were grown at 30°C in YPDA to log phase and shifted to medium containing galactose (Gal), no glucose (YP) or 1 M KCl. Cells were fixed after 15 minutes of incubation at room temperature and examined by immunofluorescence. (A) Localization of Lsm7-13myc (red in merge); P-bodies indicated by arrows. (B) Localization of Lsm8-13myc (red in merge). (C) To measure the decay rate of Lsm7p, extract from AEMY35 (PGAL1-HA-LSM7) cells harvested at 1, 2, 4, 6, 8 and 10 hours after shifting from galactose to glucose medium was tested by western blot analysis with anti-HA antibodies. (D) BMA38a with pMPS2 (GFP-Lhp1) was grown in SD-Ura-Met to log phase and shifted to 1 M KCl. Cells were fixed before and after 15 minutes in 1 M KCl. GFP-Lhp1 is shown in green; DAPI in red.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Lsm-protein delocalization upon osmotic shock is reversible and Lsm1p-independent. (A) MPS3 (Lsm8-13myc) cells grown in YPDA were fixed before and at 5, 15, 30, 60 or 120 minutes after shifting to YPDA with 0.6 M NaCl. Lsm8-13myc is shown in red; DAPI in green. (B) MRY83 (lsm1 LSM8-13myc) cells grown in YPDA were fixed before and at 5, 15 or 120 minutes after shifting to YPDA with 0.6 M NaCl. Lsm8-13myc is shown in red; DAPI in green.