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First published online 10 February 2009
doi: 10.1242/jcs.036012

Journal of Cell Science 122, 625-635 (2009)
Published by The Company of Biologists 2009
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A signal comprising a basic cluster and an amphipathic {alpha}-helix interacts with lipids and is required for the transport of Ist2 to the yeast cortical ER

Kiran Maass1,*, Marcel André Fischer1,*, Markus Seiler2, Koen Temmerman3, Walter Nickel3 and Matthias Seedorf1,{ddagger}

1 Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany
2 European Molecular Biology Laboratory (EMBL) Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
3 Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany


Figure 1
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  		Fig. 1. The sorting of Ist2 starts with rapid accumulation at the cortical ER. (A) Fluorescence of GAL1-controlled YFP-Ist2 in living ist2{Delta} cells at 60, 90 and 120 minutes after galactose induction in YEAP medium at 25°C. Scale bar: 5 µm. (B) Western blot of total extract from 0.2 OD600 ist2{Delta} cells grown for the indicated time in 2% galactose at 25°C expressing HA-Ist2 under control of the GAL1 promoter with antibodies against HA and tubulin (Tub2). (C) Immunofluorescence of cells expressing HA-Ist2 at different stages of the cell cycle after 40 minutes of galactose induction at 25°C. Nuclei are stained with DAPI. (D) Quantification of the percentage of cells (mean ± s.d.) with a detectable perinuclear HA-Ist2 signal after 40 minutes of galactose induction at 25°C. For the indicated times, 100 µg/ml cycloheximide was added to the galactose-induced cultures before fixation with formaldehyde. The percentage of cells with perinuclear HA-Ist2 (pER) in unbudded cells is shown in blue and in budded cells in red.

 

Figure 2
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  		Fig. 2. Features of the Ist2 protein. (A) Schematic overview of features of the Ist2 protein from S. cerevisiae. The transmembrane domains (1-8), the position of the DUF590 domain and the cytosol-orientated Ist2 sorting signal are shown. (B) Alignment of the Ist2 sorting signal (residues 878-946 of S. cerevisiae) with C-termini of Ist2 homologues from a selection of related yeast species. The residues in bold on top of the S. cerevisiae sequence indicate single mutations that cause mislocalisation of Ist2. The conserved positions are shown in shaded boxes.

 

Figure 3
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  		Fig. 3. Mutation of certain basic residues traps Ist2 at the perinuclear ER. (A) Localisation of YFP-tagged wild-type Ist2 and a C-terminal deletion (1-928) of YFP-Ist2 in sec23-1 cells after 120 minutes of induction with galactose at 25°C and 37°C with coexpression of GAL1-induced Hxt1-CFP in sec23-1 cells. (B) Localisation of the indicated YFP-tagged Ist2 mutants in sec23-1 cells after 120 minutes of induction with galactose at 25°C and 37°C. The mutated residues are indicated in shaded boxes. (C) Localisation of YFP-Ist2 and YFP-Ist2 basic-N in ist2{Delta} cells 120 minutes after addition of galactose and further incubation for 3 hours in glucose (Glc) at 25°C. (D) Quantification of Ist2 expression shown in C: total proteins from 0.1 OD600 unit cells were separated by SDS-PAGE and analysed by western blotting with antibodies recognising Ist2 or Sec61.

 

Figure 4
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  		Fig. 4. Mutation of the Ist2 sorting signal causes trapping at ER domains. Localisation of GAL1-induced wild-type mCherry-Ist2 or mCherry-Ist2 L942Q (in red) in strains coexpressing either GFP-tagged Scs2, Dpm1, Sec63, Hmg1, Erg6 or Sec13 (in green) in ist2{Delta} cells after induction with galactose at 25°C for 120 minutes. Scale bar: 5 µm.

 

Figure 5
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  		Fig. 5. Hydrophobic residues of the Ist2 sorting signal function as an amphipathic {alpha}-helix. (A) Presentation of residues K936-L946 as helical wheel projection. Hydrophobic leucines are marked in yellow, basic residues in blue, glycine in green and inserted alanines in grey. Depicted are wild-type Ist2, a mutant with an insertion of three alanines after position L938 and a mutant with insertion of four alanines after position G937. (B) Ist2 mutants with an exchange in the order of the leucine residues resulting in LxxLL and with an insertion of one or two alanines after position K941. The expression of the constructs in A and B was induced in sec23-1 cells for 120 minutes with galactose at 25°C or 37°C. (C,D) Secondary structure determination of the Ist2 933-946 peptide (C) and Ist2 L939P 933-946 peptide (D) by CD in 5 mM potassium phosphate buffer or in 20%, 40% and 50% TFE buffered with 5 mM potassium phosphate, respectively. The given spectra are the means of five measurements.

 

Figure 6
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  		Fig. 6. The sorting signal of Ist2 binds to peripheral yeast membranes. (A) Localisation of GFP-Ist2, GFP and GFP-Ist2 (878-946) under control of the IST2 promoter in ist2{Delta} cells. (B) Localisation of GAL1-induced GFP-Ist2 (878-946) in ist2{Delta} cells after addition of galactose for 3 hours and further incubation for 8 hours in glucose. (C) Supernatant and 25,000 g membrane pellet of ist2{Delta} cells expressing GFP or GFP-Ist2 (878-946) under the control of the IST2 promoter. Proteins from 1 OD600 unit supernatant (S) and pellet (P) were analysed by western blotting with antibodies against GFP, G6PDH and Sec61.

 

Figure 7
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  		Fig. 7. The sorting signal of Ist2 binds to lipids. (A) Pellet fraction of 25,000 g membranes of ist2{Delta} cells was treated either with buffer or proteinase K and incubated with recombinant GFP or GFP-Ist2 (878-946). The reactions were separated into supernatant and pellet fraction, analysed by western blotting with antibodies against GFP and Sec61. (B) Binding of recombinant GFP and GFP-Ist2 (878-946) to liposomes. Liposomes were incubated with 15 µg recombinant protein in 100 µl buffer and fractionated into supernatant and pellet. (C) Binding of recombinant GFP, GFP-Ist2 (878-946) and different mutants of GFP-Ist2 (878-946) to liposomes. Liposomes were incubated with 15 µg recombinant protein in 100 µl buffer, sorted via flow cytometry and GFP fluorescence was detected. The relative GFP fluorescence of GFP-Ist2 (878-946) bound to liposomes was set to 100. Values are means ± s.d.

 

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© The Company of Biologists Ltd 2009