spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/jcs.00189

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jensen, S.
Right arrow Articles by Johnston, L. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jensen, S.
Right arrow Articles by Johnston, L. H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae

Sanne Jensen1, Marco Geymonat1, Anthony L. Johnson1, Marisa Segal2 and Leland H. Johnston1,*

1 Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK



View larger version (41K):

[in a new window]
  		Fig. 1. Mitotic Lte1 localisation is actin independent but requires activation of Cdc28/Cln kinase. (A) cln1{Delta}cln2{Delta}cln3{Delta} GAL1-CLN3 strain (SJ123) was arrested in G1 and released into YEPGalactose to induce expression of Cln3 at 30°C. Aliquots were removed at indicated times for analysis by real time microscopy, determination of budding index and cell cycle progression by DAPI stain. ({square}) Budding index; ({diamondsuit}) cortical Lte1; ({circ}) cells with pre-anaphase nuclear morphology; ({triangleup}) cells with anaphase nuclear morphology; (•) cells with completely separated DNA masses (telophase). (B) Cells described in (A) were arrested in G1 (a) and released into the cell cycle in the presence of LatB (b) or DMSO as control (c). Localisation of Lte1GFP monitored after 2 hours. The percentage of cells with cortical Lte1 is indicated below. (C) Wild-type cells (SY125) were arrested in G1 with {alpha}-factor (a). Shmoos were treated with LatB for 15 minutes and cells examined by microscopy (b-c). Numbers indicate the percentage of cells that localised Lte1GFP to the tip of the mating projection. (D) Localisation of Lte1GFP in cdc15-1 mutant cells expressing CFP-tubulin (SY126) at the restrictive temperature (a), following 1 hour nocodazole treatment at 37°C (b) and 1 hour after release at permissive temperature (c). Numbers indicate the percentage of cells with cortical Lte1. Bar, 10 µm.

 


View larger version (41K):

[in a new window]
  		Fig. 2. Lte1 cortex association is largely independent of an intact secretion pathway but responds to defects in septin structure. (A,B) Wild-type and sec18-1 (SY132) cells were shifted to 37°C for 90 minutes and Lte1 localisation was observed and quantified. Bar, 10 µm. (C) Lte1GFP localisation in wild-type and cdc12-6 (SY134) mutant cells at permissive temperature and following 20 minutes shift to 37°C. (D) Introduction of CDC12, but not plasmid alone, restores Lte1 to the cortex (a-b). (E) Phosphorylation status/stability of Lte1 is not affected by septin mutation. Wild-type and cdc12-6 (SY133) strains expressing HA3 tagged Lte1 were arrested with nocodazole at 20°C. Cultures were shifted 20 minutes to 37°C and cells collected for protein analysis.

 


View larger version (39K):

[in a new window]
  		Fig. 3. Distribution of Lte1 in polarity mutants. (A) Localisation of Lte1GFP in a cdc28-13 strain (SY127) at the permissive temperature and after incubation at 37°C. (B) Haploid cdc42-1 cells expressing either endogenous Lte1GFP (SY128, upper panel) or Cdc24GFP (SY129, bottom panel) were examined at the permissive temperature and 2 hours after shift to 37°C. (C) Haploid gic1{Delta}gic2{Delta} cells expressing endogenous Lte1GFP (SY151) were shifted to 37°C for 3 hours and Lte1 localisation monitored by microscopy. (D) Wild-type (SY125), bni1{Delta} (SY130) and bud6{Delta} (SY131) cells were released from their block in G0 at 30°C and analysed for Lte1 localisation after 2 hours. (A-D) Numbers indicate the percentage of cells with shown localisation pattern.

 


View larger version (29K):

[in a new window]
  		Fig. 4. Cdc14 activity induces dissociation of Lte1 from the cortex. (A) A strain carrying GAL1-inducible CDC14 (SY140) was grown in YPRaffinose and arrested with nocodazole. At the arrest either 2% galactose (+) or 2% dextrose (-) was added. At the indicated times Lte1GFP fluorescence was monitored. Representative cells are shown below the table. Bar, 10 µm. (B) A strain harbouring a GAL1-inducible SIC1 plasmid (SY139) was treated as described in A. Cells were quantified in a similar fashion. A multibudded phenotype was observed upon extensive SIC1 overexpression but pictures are representative of cells at the 2 hour time point.

 


View larger version (28K):

[in a new window]
  		Fig. 5. The Cdc14 phosphatase dephosphorylates Lte1 in vivo and in vitro. (A) A strain expressing endogenous HA3-tagged Lte1 and carrying a GAL1-inducible CDC14 plasmid (SY138) was treated as described in Fig. 4A. At the indicated times, samples were withdrawn for protein analysis, Clb2-associated kinase assays using Histone H1 as substrate and FACS analysis. The arrow indicates position of unphosphorylated Lte1. (B) Similar analysis to that described in A was performed on a strain expressing Lte1HA3 and carrying a GAL1-inducible SIC1 plasmid (SY137). Multiple Lte1 forms are indicated with a bracket. An unspecific band seen with the anti-Sic1 antibody is indicated with an asterisk (C). Extracts from nocodazole-arrested cells expressing endogenous Lte1 HA3 tagged protein were subjected to immunoprecipitation using anti-HA antibody. The immunoprecipitates were left untreated (lane 1), incubated with 1 µg purified MBP-Cdc14 (lane 2), 1 µg MBP-Cdc14C283A (lane 3) or 1 µg MBP (lane 4).

 


View larger version (57K):

[in a new window]
  		Fig. 6. Lte1 is phosphorylated by Cdc28 across the cell cycle. (A) Wild-type cells (SY142) were released from an {alpha}-factor-induced G1 block into fresh YPDextrose medium at 25°C. Cells were collected at indicated intervals and processed for immunoblotting. The asterisk indicates the position of an unspecific band. (B) Cells containing a non-degradable version of Sic1 under the control of the GAL promoter (SY135) were synchronised in G1 with {alpha}-factor. Cells were released into either YPSucrose or YPGalactose medium at 30°C. At 15 minute intervals, samples were withdrawn for western blotting to monitor Lte1 phosphorylation and FACS analysis. Cells expressing Lte1 GFP (SY136) were treated in a similar fashion and the effect of Sic1{Delta}N overproduction on Lte1 localisation scored by microscopy (lower panel). Numbers indicate the percentage of cells with cortical Lte1. (C) Left panel, GST-Cdc28-13 purified from asynchronous yeast cells was used for kinase assays at 25°C and 37°C with either GST-Lte1(300-845) or Histone H1 as substrates. The Cdc28-13 kinase was pre-incubated at 37°C for 30 minutes prior to use in reactions performed at 37°C. Middle panel, GST-Cdc28/Cln2 complexes purified on glutathione sepharose beads from cdc53-1 yeast cells (SY141) arrested at the restrictive temperature were used for kinase assays with either GST-Lte1(300-845) or Histone H1 as substrates. Extract from uninduced cdc53-1 cells was used as a negative control. Right panel, purified GST-Lte1(300-845) and GST-Lte1Cdk(300-845) proteins were used in kinase assays with Cdc28 purified from nocodazole-treated yeast cells. The band of radioactive protein migrated to the same position as the Lte1 protein detected by Coomassie staining (input). (D) Cells expressing Lte1CdkHA3 (SY143) were synchronised in G1 by {alpha}-factor and released into YPDextrose medium at 25°C. Cells were then treated as in A. (E) Localisation of Lte1GFP in G1 cyclin-depleted cells (SY124) (—Cln2), after 3 hours overexpression of Cdc42-G12V from the GAL promoter (—Cln2+galactose). The number indicates cells with clear cytoplasmic staining. Cells released from the arrest localised Lte1 efficiently at the cortex (+Cln2). Here the number indicates cells with cortical distribution.

 


View larger version (33K):

[in a new window]
  		Fig. 7. Functional domain analysis of Lte1. (A) A schematic representation of the domain structure of Lte1 is shown. The N-terminal region contains a GEFN motif, whereas the Cdc25-related GEF domain resides in the extreme C-terminus. Mutants were either expressed from an integrated allele driven by the GAL1 promoter (GAL1) or from an integrated allele driven by the LTE1 promoter (endogenous). Complementation was scored as the ability to rescue the growth defect of lte1{Delta} mutant (SY144) by serial dilution spot analysis after 7 days incubation at 14°C. Lte1 mutants were tested for complementation with or without GFP/HA3 tag with roughly similar outcome. +, cortical localisation; -, cytoplasmic/none localisation; +/-, partial cortical localisation; ND, not determined. (B) Representative cells of Lte1 mutants expressed either from GAL1 promoter or at endogenous level; Lte1{Delta}C984GFP (SY149); Lte1{Delta}N801GFP (SY150); wild-type Lte1GFP (SY148); Lte1{Delta}N157GFP (SY146); Lte1{Delta}C1334GFP (SY147); wild-type Lte1GFP (SY145). (C) Equal amounts of GST-Lte1{Delta}N984 protein or GST were bound to glutathione sepharose beads and incubated with MBP-Lte1 (1-500). Bound protein was eluted with glutathione and analysed by SDS-PAGE (10% gels) and immunostaining with anti-MBP antibody.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002