Figure 7.
Epistatic relation of proteins at LD biogenesis sites. (A) Lack of FIT proteins does not abrogate recruitment of GFP-FFAT-sLro1 at Fld1 sites. yft2Δ scs3Δ double mutant cells expressing Fld1-mCherry and coexpressing GFP-FFAT-sLro1 were switched to galactose media for 1 h. White arrowheads indicate colocalization of Fld1 with sLro1. (B) Cells lacking FIT proteins have elevated DAG levels at Fld1 sites. WT and yft2Δ scs3Δ double mutant cells expressing Fld1-mCherry and coexpressing the GFP-tagged ER-DAG sensor were grown to early stationary phase. Arrowheads indicate colocalization of Fld1 foci with the DAG-sensor punctae. (C) Cartoon showing accumulation of DAG at Fld1- and Nem1-marked ER subdomains when these sites are missing Yft2. (D and E) Pex30 acts downstream of Fld1 and Nem1 in recruiting GFP-FFAT-sLro1 to sites of LD biogenesis. pex30Δ mutant cells expressing Fld1-mCherry (D) or Nem1-mCherry (E) and coexpressing GFP-FFAT-sLro1 were switched to galactose-containing media for the indicated time. (F) Yft2 functions independently of Pex30 at sites of LD biogenesis. pex30Δ mutant cells expressing Fld1-mCherry or Nem1-mCherry and coexpressing Yft2-sf-GFP were diluted into fresh SC media containing 0.1% OA and imaged after 1 h. White arrowheads indicate colocalization of Yft2 with Fld1 or Nem1 punctae. (G and H) Cartoon illustrating the localization of Fld1, Nem1, Yft2, and Pex30 at ER subdomains to constitute functional LD biogenesis sites (G). Absence of Pex30 from these sites does not affect the localization of Fld1, Nem1, and Yft2, but these sites fail to recruit the sLro1 (H). (I) Lack of Pet10 does not impair the recruitment of GFP-FFAT-sLro1 at Fld1 sites. pet10Δ mutant cells expressing Fld1-mCherry and coexpressing GFP-FFAT-sLro1 were grown as described in D. Arrowheads denote colocalization of sLro1 with Fld1 foci. Scale bar, 5 µm. (J) Cartoon illustrating localization of sLro1 at Fld1 sites in the absence of Pet10.

Epistatic relation of proteins at LD biogenesis sites. (A) Lack of FIT proteins does not abrogate recruitment of GFP-FFAT-sLro1 at Fld1 sites. yft2Δ scs3Δ double mutant cells expressing Fld1-mCherry and coexpressing GFP-FFAT-sLro1 were switched to galactose media for 1 h. White arrowheads indicate colocalization of Fld1 with sLro1. (B) Cells lacking FIT proteins have elevated DAG levels at Fld1 sites. WT and yft2Δ scs3Δ double mutant cells expressing Fld1-mCherry and coexpressing the GFP-tagged ER-DAG sensor were grown to early stationary phase. Arrowheads indicate colocalization of Fld1 foci with the DAG-sensor punctae. (C) Cartoon showing accumulation of DAG at Fld1- and Nem1-marked ER subdomains when these sites are missing Yft2. (D and E) Pex30 acts downstream of Fld1 and Nem1 in recruiting GFP-FFAT-sLro1 to sites of LD biogenesis. pex30Δ mutant cells expressing Fld1-mCherry (D) or Nem1-mCherry (E) and coexpressing GFP-FFAT-sLro1 were switched to galactose-containing media for the indicated time. (F) Yft2 functions independently of Pex30 at sites of LD biogenesis. pex30Δ mutant cells expressing Fld1-mCherry or Nem1-mCherry and coexpressing Yft2-sf-GFP were diluted into fresh SC media containing 0.1% OA and imaged after 1 h. White arrowheads indicate colocalization of Yft2 with Fld1 or Nem1 punctae. (G and H) Cartoon illustrating the localization of Fld1, Nem1, Yft2, and Pex30 at ER subdomains to constitute functional LD biogenesis sites (G). Absence of Pex30 from these sites does not affect the localization of Fld1, Nem1, and Yft2, but these sites fail to recruit the sLro1 (H). (I) Lack of Pet10 does not impair the recruitment of GFP-FFAT-sLro1 at Fld1 sites. pet10Δ mutant cells expressing Fld1-mCherry and coexpressing GFP-FFAT-sLro1 were grown as described in D. Arrowheads denote colocalization of sLro1 with Fld1 foci. Scale bar, 5 µm. (J) Cartoon illustrating localization of sLro1 at Fld1 sites in the absence of Pet10.

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