Figure 4.
The N-terminal amphipathic helix of Svf1 targets to the ER. (a) Graphical representation of the AH of Svf1 (yellow; WT, left; V12D mutant, middle; G7A/G8A mutant, right) tagged with GFP. (b) Co-localization of the GFP tagged AH expressed from a plasmid under the control of the promoter of Svf1 (WT, upper panel; V12D, lower panel) with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (c) Western blot analysis of the WT and the V12D AH (WT and AHV12D, respectively) fused to GFP. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the localization of the GFP tagged AH (WT) and AHV12D 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. (d) Co-localization of the GFP tagged AH expressed from a plasmid under the control of the endogenous promoter of Svf1 (WT, upper panel; G7A/G8A, lower panel) with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (e) Western blot analysis of the WT and the G7A/G8A AH (AHWT and AHG7A/G8A, respectively) fused to GFP. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the localization of the GFP tagged AHWT and AHG7A/G8A 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 and g) Helical wheel representation of the 1–18 amino acids of Grh1 as described for Svf1 in Fig. 3 a and (g) represented in the model in purple (right), which were fused to Svf1 (19–481 aa; orange) and a C-terminal GFP tag (green). (h) Co-localization of GFP tagged Svf1 expressed from a plasmid under the control of the endogenous promoter of Svf1 with mKate tagged Mnn9 (upper panel) and the fused protein with the AH of Grh1 (1–18 aa) and Svf1 (19–481 aa) expressed from a plasmid under control of the endogenous promoter of Svf1 with mKate tagged Mnn9 (lower panel). Scale bar = 5 µM. Source data are available for this figure: SourceData F4.

The N-terminal amphipathic helix of Svf1 targets to the ER. (a) Graphical representation of the AH of Svf1 (yellow; WT, left; V12D mutant, middle; G7A/G8A mutant, right) tagged with GFP. (b) Co-localization of the GFP tagged AH expressed from a plasmid under the control of the promoter of Svf1 (WT, upper panel; V12D, lower panel) with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (c) Western blot analysis of the WT and the V12D AH (WT and AHV12D, respectively) fused to GFP. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the localization of the GFP tagged AH (WT) and AHV12D 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. (d) Co-localization of the GFP tagged AH expressed from a plasmid under the control of the endogenous promoter of Svf1 (WT, upper panel; G7A/G8A, lower panel) with the organelle markers Mnn9-mKate (cis-Golgi) and Sec63-Halo (ER). Scale bar = 5 µM. (e) Western blot analysis of the WT and the G7A/G8A AH (AHWT and AHG7A/G8A, respectively) fused to GFP. The separation of the cell lysate (Input, I) into pellet (P) and supernatant (S) fractions shows the localization of the GFP tagged AHWT and AHG7A/G8A 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 and g) Helical wheel representation of the 1–18 amino acids of Grh1 as described for Svf1 in Fig. 3 a and (g) represented in the model in purple (right), which were fused to Svf1 (19–481 aa; orange) and a C-terminal GFP tag (green). (h) Co-localization of GFP tagged Svf1 expressed from a plasmid under the control of the endogenous promoter of Svf1 with mKate tagged Mnn9 (upper panel) and the fused protein with the AH of Grh1 (1–18 aa) and Svf1 (19–481 aa) expressed from a plasmid under control of the endogenous promoter of Svf1 with mKate tagged Mnn9 (lower panel). Scale bar = 5 µM. Source data are available for this figure: SourceData F4.

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