Figure 3.
Svf1 possesses an N-terminal amphipathic helix important for the targeting and function of the protein. (a and b) Helical wheel representation of the first 18 amino acids with the hydrophobic amino acids shown in yellow and the hydrophobic moment shown by the arrow and expressed in µH above the arrow for the WT sequence and (b) the V12D mutant with the exchange of the hydrophobic valine to the charged aspartate (red). (c) Graphical representation of the full-length protein (WT, left; V12D mutant, right). Shown are the AH (yellow, 1–18 aa), the rest of Svf1 (orange, 19–481 aa) and the C-terminal GFP tag (green). (d) Co-localization of GFP tagged Svf1 expressed from a plasmid under control of the endogenous promoter with mCherry tagged Mnn9 for the WT (upper panel) and the V12D mutant (lower panel). Scale bar = 5 µM. Scale bar inlays = 1 µM. (e) Samples from the membrane fractionation according to 50 µg protein concentration were analyzed by Western blot. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the Svf1 localization either bound to membranes (P) or cytosolic (S) detected by an α-GFP antibody. The antibodies α-Pgk1 and α-Sec61 were used as loading controls for the cytosol and membrane fractions, respectively. (f) Tetrad analysis of the svf1Δpl.Svf1-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). (g) Tetrad analysis of the svf1Δpl.Svf1V12D-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). (h) A model of Svf1 with the folded (bound Svf1) and unfolded (cyt. Svf1) N-terminal AH. (i) Tetrad analysis of the svf1∆pl.Svf1L2E-GFP (blue and green, respectively) mutants crossed with sur2∆ (red). (j) Western blot analysis of Svf1-GFP in WT and mak3Δ mutant as described in e. (k and l) Helical wheel representation of the WT AH (k) and the G7A/G8A mutant AH (l) described as in Fig. 3 a shows no impact by the exchanges of glycines to alanines at the positions 7 and 8 on the hydrophobic moment of the AHs. (m) Co-localization of GFP tagged Svf1 expressed from a plasmid under the control of the endogenous promoter of Svf1 in a svf1Δ background (WT, upper panel; G7A/G8A mutant, lower panel), with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (n) Western blot analysis as described in e for Svf1WT-GFP and Svf1G7A/G8A-GFP. Source data are available for this figure: SourceData F3.

Svf1 possesses an N-terminal amphipathic helix important for the targeting and function of the protein. (a and b) Helical wheel representation of the first 18 amino acids with the hydrophobic amino acids shown in yellow and the hydrophobic moment shown by the arrow and expressed in µH above the arrow for the WT sequence and (b) the V12D mutant with the exchange of the hydrophobic valine to the charged aspartate (red). (c) Graphical representation of the full-length protein (WT, left; V12D mutant, right). Shown are the AH (yellow, 1–18 aa), the rest of Svf1 (orange, 19–481 aa) and the C-terminal GFP tag (green). (d) Co-localization of GFP tagged Svf1 expressed from a plasmid under control of the endogenous promoter with mCherry tagged Mnn9 for the WT (upper panel) and the V12D mutant (lower panel). Scale bar = 5 µM. Scale bar inlays = 1 µM. (e) Samples from the membrane fractionation according to 50 µg protein concentration were analyzed by Western blot. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the Svf1 localization either bound to membranes (P) or cytosolic (S) detected by an α-GFP antibody. The antibodies α-Pgk1 and α-Sec61 were used as loading controls for the cytosol and membrane fractions, respectively. (f) Tetrad analysis of the svf1Δpl.Svf1-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). (g) Tetrad analysis of the svf1Δpl.Svf1V12D-GFP (blue and green, respectively) mutants crossed with sur2Δ (red). (h) A model of Svf1 with the folded (bound Svf1) and unfolded (cyt. Svf1) N-terminal AH. (i) Tetrad analysis of the svf1∆pl.Svf1L2E-GFP (blue and green, respectively) mutants crossed with sur2∆ (red). (j) Western blot analysis of Svf1-GFP in WT and mak3Δ mutant as described in e. (k and l) Helical wheel representation of the WT AH (k) and the G7A/G8A mutant AH (l) described as in Fig. 3 a shows no impact by the exchanges of glycines to alanines at the positions 7 and 8 on the hydrophobic moment of the AHs. (m) Co-localization of GFP tagged Svf1 expressed from a plasmid under the control of the endogenous promoter of Svf1 in a svf1Δ background (WT, upper panel; G7A/G8A mutant, lower panel), with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (n) Western blot analysis as described in e for Svf1WT-GFP and Svf1G7A/G8A-GFP. Source data are available for this figure: SourceData F3.

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