A signal comprising a basic cluster and an amphipathic -helix interacts with lipids and is required for the transport of Ist2 to the yeast cortical ER

JCS036012 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. YFP-Ist2 can be detected at the perinuclear ER by immunofluorescence. Immunofluorescence of HA-Ist2- and YFP-Ist2-expressing cells after 40 minutes of galactose induction at 25°C. DNA is stained with DAPI. HA-Ist2 and YFP-Ist2 were detected using an Ist2 antibody. The arrows indicate accumulation at the perinuclear ER.

• Supplemental Figure S10 -

  Fig. S10. Ist2 L942Q induces expression of Kar2. (A) Western blot of total extract from 0.25 OD₆₀₀ ist2Δ cells expressing GAL1-induced YFP-Ist2 L942Q, YFP-Ist2 for 3 hours and ist2Δ cells treated with 5 mM DTT for 3 hours. All experiments were performed at 25°C, proteins were detected using antibodies specific for Ist2, Kar2 and Sec61. (B) Quantification of three independent experiments performed as in (A). The Kar2 signal after induction of the cells was quantified, normalized with the Sec61 loading control and divided by the normalized Kar2 signal before induction of the cells. Shown is the mean of three experiments. Error bars indicate the s.d. (C) Immunofluorescence of ist2Δ cells expressing mCherry-Ist2 L942Q after 2 hours of galactose induction at 25°C. mCherry-Ist2 L942Q was detected via mCherry fluorescence, Kar2 was detected with a specific antibody, DNA is stained with DAPI. The arrow indicates colocalisation of Kar2 and Ist2 L942Q. (D) Quantification of unbudded cells and daughter cells showing colocalisation of mCherry-Ist2 L942Q and Kar2.

• Supplemental Figure S11 -

  Fig. S11. Temperature dependent accumulation of Ist2 L942V. Localisation of wild-type YFP-Ist2 and mutant YFP-Ist2 L942V in ist2Δ cells. After 120 minutes of induction with galactose at 25°C, the culture was divided and further incubated for 1 hour with glucose (Glc) either at 25°C or 37°C. Quantification of the percentage of cells with YFP-Ist2 L942V dots (in at least 100 cells) after induction for 120 minutes with galactose at 25°C followed by incubation at 37°C in medium containing glucose and 10 mM sodium azide (filled circles) or in medium with glucose (filled triangles).

• Supplemental Figure S12 -

  Fig. S12. Insertion of a proline into the amphipathic α-helix abolishes function of the Ist2 sorting signal. Localisation of YFP-Ist2 L939P at 25° or 37°C in sec23-1 cells after induction for 120 minutes with galactose.

• Supplemental Figure S13 -

  Fig. S13. Ist2 878-946 redirects a lumenal and membrane-anchored YFP from the perinuclear ER to the cell periphery. Localisation of a constructs containing a lumenal YFP followed by the first transmembrane domain of Sec63 in ist2D cells at 25°C for 120 minutes after induction with galactose. SS-YFP-TM indicates the construct with a stop codon after the transmembrane domain and SS-YFP-TM-Ist2 (878-946), the construct with Ist2 (878-946) at its C-terminus. The arrows indicate localisation at the perinuclear ER.

• Supplemental Figure S14 -

  Fig. S14. GFP-Ist2 (878-946) floats with yeast membranes and binds to membranes in a salt-dependent manner. (A) 100 OD₆₀₀ ist2Δ cells expressing GFP-Ist2 (878-946) under the control of the IST2 promoter were fractionated into supernatant and 25,000 g pellet. The entire pellet fraction was resuspended in 2 M sucrose and overlaid with a sucrose step gradient. After ultracentrifugation, equal fractions were collected from top to bottom and analysed by western blotting with antibodies against GFP and Sec61. (B) ist2Δ cells expressing GFP-Ist2 (878-946) under the control of the IST2 promoter were fractionated into supernatant and pellet as in Fig. 6C using increasing concentrations of potassium acetate in yeast lysis buffer.

• Supplemental Table S1 -

• Supplemental Table S2 -

• Supplemental Table S3 -

• Supplemental Figure S2 -

  Fig. S2. Lys936 has a special role in trafficking of Ist2. (A) Localisation of GAL1-induced YFP-tagged Ist2 mutants K935A, K936A and K933A/H934A. (B) Localisation of GAL1-induced YFP-tagged Ist2 K936, H940, K941, K943, K944, K945 to A (K936A/basic-C). The mutated residues are indicated in shaded boxes. Expression of all constructs was induced for 120 minutes with galactose at 25° or 37°C in sec23-1 cells.

• Supplemental Figure S3 -

  Fig. S3. Function of lysine residues N-terminal of Lys936. (A) Localisation of GAL1-induced YFP-tagged Ist2 mutants K931A/K933A/K934A, K931A/H934A/K935A, K931A/K933A/K935A and K933A/H934A/K935A. (B) Localisation of YFP-tagged Ist2 K931A/K933A/H934A/K935A. The mutated residues are indicated in shaded boxes. Expression of all constructs was induced for 120 minutes with galactose at 25°C or 37°C in sec23-1 cells.

• Supplemental Figure S4 -

  Fig. S4. The expression level effects the localization of YFP-Ist2 L942Q. (A) Expression of YFP-Ist2 (lane 1) and two different YFP-Ist2 L942Q clones (low and high expression, lanes 2 and 3) in sec23-1 cells after 120 minutes galactose induction. Total proteins from 0.1 OD₆₀₀ cells grown at 25° or 37°C were analysed by western blotting with antibodies recognizing Ist2 or Sec61. (B) Localisation of wild-type YFP-Ist2, YFP-Ist2 L942Q low and YFP-Ist2 L942Q high after 120 minutes galactose induction at 25° or 37°C with coexpression of GAL1-induced Hxt1-CFP in sec23-1 cells.

• Supplemental Figure S5 -

  Fig. S5. Position 942 requires hydrophobic and bulky residues. Localisation of the indicated YFP-Ist2 L942 mutants in sec23-1 cells after 120 minutes in galactose at 25°C or 37°C.

• Supplemental Figure S6 -

  Fig. S6. Hydrophobicity of residues L938 and L939 contributes to the function of the Ist2 sorting signal. Localisation of YFP-Ist2 L938A, L938V, L983I, L939A, L939V, L939I, L938A/L939A, L938V/L939V, L938I/L939I, L946A, L946V, L946I mutants in sec23-1 cells after 120 minutes in galactose at 25°C or 37°C.

• Supplemental Figure S7 -

  Fig. S7. Mutant Ist2 does not colocalise with Golgi or endosomes. Localisation of GAL1-induced wild-type GFP-Ist2 and mutant GFP-Ist2 L942Q (in green) in ist2Δ cells that coexpress Sec7-RFP (in red) and localisation of GAL1-induced wild type mCherry-Ist2 and mutant mCherry-Ist2 L942Q (in red) in ist2Δ cells that coexpress GFP-tagged Sed5, Gef1, or Kex2 (in green) after 120 minutes at 25°C. Scale bar: 5 µm.

• Supplemental Figure S8 -

  Fig. S8. Statistical analysis of colocalisation. The distance of the maximum signal intensity of an Ist2 L942Q dot and the respective markers was determined in pixel. 5 pixel equal 0.4 µm. (A) Colocalisation with the Golgi-markers Sec7, Sed5, Gef1 and Kex2. (B) Colocalisation with the ER-markers Scs2, Dpm1, Sec63 and Hmg1. (C) Colocalisation with Sec13 (ER-exit sites) and Erg6 (lipid droplets).

• Supplemental Figure S9 -

  Fig. S9. Ist2 L942Q cofractionates with rough ER. (A) Cell lysate of 10 OD₆₀₀ ist2Δ cells expressing Pma1-GFP and GAL1-induced mCherry-Ist2 L942Q was subjected to ultracentrifugation in a 20-60% sucrose gradient. Fractions were collected from top to bottom and analysed by western blot with Ist2, Sec61, GFP and Emp47 antibodies. (B) Quantification of the distribution of the proteins analysed in (A). (C) Subcellular fractionation of cells expressing Sed5-GFP and GAL1-induced mCherry-Ist2 L942Q as in A, and quantification of the distribution of mCherry-Ist2 L942Q, Sec61 and Sed5-GFP. (D) Subcellular fractionation of cells expressing Sec13-GFP and GAL1-induced mCherry-Ist2 L942Q as in A, and quantification of the distribution of mCherry-Ist2 L942Q, Sec61 and Sec13-GFP. (E) Cells expressing Sec13-GFP were fractionated into supernatant and 25,000 g pellet. The fractions were probed with antibodies against GFP, Sec61 and G6PDH. Quantification revealed that 65% of Sec13-GFP was found in the pellet fraction.