![Rescue of ERMES loss by Mmm1’s SMP domain is independent of orientation and requires lipid transport activity. (A) Schematic of constructs used in B. (B) Spot assay of mmm1Δmdm12Δmdm34Δmdm10Δ yeast strains expressing tethering constructs with different transmembrane and SMP domains. (C) Predicted mutant Mmm1 SMP domain structure based on Damietta and AlphaFold. The engineered mutations affecting lipid binding are indicated in pink. Phospholipids comes from the overlaid crystal structure of Zygosaccharomyces rouxii (PDB: 5YK6, [Jeong et al., 2017], RMSD = 0.806 Å). (D) Protein levels of Mmm1-mNG-Fis1C with or without mutations that impair lipid transfer activity. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. Tom70 was used as loading controls. The asterisk indicates non specific signal. (E) Spot assay of mmm1Δ mdm12Δ mdm34Δ mdm10Δ yeast cells expressing MMM1-mNG-Fis1C constructs. The wild-type construct is compared to a lipid transport–deficient quintuple mutant of the Mmm1 SMP domain. Source data are available for this figure: SourceData F5. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jcb/225/7/10.1083_jcb.202411196/1/jcb_202411196_fig5.png?Expires=1781310138&Signature=en2wImg9NX7HYCAjlMtsRsHIAxwJOiGoSoBf3p3REU8n~G9ylNfFT~bbe29BC2eqknnQGXB4cIDyWjw2YsA963W~ktDyhIKUEsOzTz7Z5Z1-pEpPTWEHOAVBC7xOgCnMlkfH6V~IwxP1fBr1NuVjyNpz4ybC7eaaMXW1POh4NQDRCRo7zqnoZ75ypDtwvz4PJQZ9MYPmiboys1gjp~LGNkrhs6O4IJsQGhexsAZMBPA8kffCedwq4C3uOEII08hfJ7f6Yi2SbbwZGOuCJnO3PbSziATr1qbosK~nH7yE307nJjgIspnRv10UNDp3aIj7IhHoYjHTZtCa8GcgT~i~jA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A: A schematic diagram of protein constructs with different transmembrane and SMP domains. The constructs include Sec66-TM, Ubc6-TM, Fis1-TM, and Tom70-TM, each labeled with mNG. Panel B: A spot assay showing the growth of yeast strains expressing different constructs. The constructs are listed on the left, and the growth patterns are shown in rows. Panel C: A structural diagram of the mutant Mmm1 SMP domain, highlighting engineered mutations. Phospholipids are overlaid from a crystal structure. Panel D: Immunoblot analysis of Mmm1-mNG-Fis1C expression levels in the presence and absence of specific mutations. Tom70 is provided as a mitochondrial loading control. Panel E: Representative micrographs illustrating the subcellular distribution of mutant Mmm1-mNG-Fis1C. Colocalization is assessed using ER and mitochondrial fluorescent markers for reference.
Rescue of ERMES loss by Mmm1’s SMP domain is independent of orientation and requires lipid transport activity. (A) Schematic of constructs used in B. (B) Spot assay of mmm1Δmdm12Δmdm34Δmdm10Δ yeast strains expressing tethering constructs with different transmembrane and SMP domains. (C) Predicted mutant Mmm1 SMP domain structure based on Damietta and AlphaFold. The engineered mutations affecting lipid binding are indicated in pink. Phospholipids comes from the overlaid crystal structure of Zygosaccharomyces rouxii (PDB: 5YK6, [Jeong et al., 2017], RMSD = 0.806 Å). (D) Protein levels of Mmm1-mNG-Fis1C with or without mutations that impair lipid transfer activity. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. Tom70 was used as loading controls. The asterisk indicates non specific signal. (E) Spot assay of mmm1Δ mdm12Δ mdm34Δ mdm10Δ yeast cells expressing MMM1-mNG-Fis1C constructs. The wild-type construct is compared to a lipid transport–deficient quintuple mutant of the Mmm1 SMP domain. Source data are available for this figure: SourceData F5.