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. 2012 Jun;13(6):880-90.
doi: 10.1111/j.1600-0854.2012.01352.x. Epub 2012 Apr 8.

Gem1 and ERMES do not directly affect phosphatidylserine transport from ER to mitochondria or mitochondrial inheritance

Tammy T Nguyen  1 , Agnieszka Lewandowska, Jae-Yeon Choi, Daniel F Markgraf, Mirco Junker, Mesut Bilgin, Christer S Ejsing, Dennis R Voelker, Tom A Rapoport, Janet M Shaw
Affiliations
Free PMC article

Gem1 and ERMES do not directly affect phosphatidylserine transport from ER to mitochondria or mitochondrial inheritance

Tammy T Nguyen et al. Traffic. 2012 Jun.
Free PMC article

Abstract

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.

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Figures

Figure 1
Figure 1
ERMES is not essential for PS transfer between the ER and mitochondria. A) The conversion of PS to PE was determined in vivo by incubating yeast cells with radioactive serine, extracting phospholipids, and analysis by TLC. The percentage of radiolabeled PS converted to PE in psd2Δ control and mutant strains is shown. Labeling of the different ERMES mutants was comparable with the exception of mdm10Δ, which consistently incorporated less label than psd2Δ control and the other mutants. B) The conversion of PS to PE was determined in vitro using crude mitochondria incubated with radioactive serine. The percentage of radiolabeled PS converted to PE in WT and mutant mitochondria is shown. Bars and error bars represent the average and SD from three independent experiments. C) Relative changes in phospholipid composition in ERMES mutants. Lipid extracts of the indicated strains were analyzed by shotgun lipidomics . The abundance of the indicated lipid classes are shown relative to the control strain psd2Δ. Samples were analyzed in duplicate and average values are shown. The maximal difference between two duplicate measurements was 1.2%. PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol.
Figure 2
Figure 2
Deletion of GEM1 does not alter cellular and mitochondrial phospholipid profiles, or the transport dependent conversion of PS to PE. A) Transport of PS and conversion into PE was assayed in vivo by growing the indicated strains in medium containing [3H]-serine and analyzing the distribution of radioactivity among phospholipid classes. (B) Synthesis and transport of PS and conversion into PE was measured in vitro by pulse labeling the crude mitochondrial fraction with [3H]-serine for 15 minutes to radiolabel the PS pool, and then arresting further PS synthesis and following the conversion to [3H]-PE. The reactions were terminated by lipid extraction and the distribution of radioactivity among phospholipid classes was quantified. To assess steady state lipid content, lipids were also extracted from whole cells (C), or mitochondria isolated from psd2Δ and gem1Δ psd2Δ strains (D). Individual lipid classes were separated by two-dimensional TLC and quantified by measuring phosphorus. Bars and error bars represent the average and SD from three independent experiments. CL, cardiolipin; PA, phosphatidic acid. Other lipid abbreviations as in Figure 1.
Figure 3
Figure 3
Mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects. Quantification of mitochondrial inheritance (A) and (B) or mitochondrial morphology (C) in strains expressing a fluorescent mitochondrial marker. Genotypes of strains containing vector alone or expressing Ypt11 or ChiMERA are indicated. Bars and error bars represent the average and SD from three independent experiments.
Figure 4
Figure 4
Expression of ChiMERA, but not Ypt11, rescues growth defects caused by deletion of MDM12. mdm12Δ (top) containing vector alone (vector), the synthetic ER-mitochondria tether (ChiMERA), or YPT11 were grown on synthetic dextrose (SD) selective medium for 3 days at 30 °C. Neither ChiMERA nor YPT11 expression rescued growth defects in mdm10Δ (middle) or mmm1Δ (bottom).
Figure 5
Figure 5
ERMES puncta formation does not require GEM1 or genes implicated in mitochondrial inheritance. Differential interference contrast (DIC, left), Mmm1-GFP (middle), and Mmm1-GFP merged with mtRFP (right) images of the indicated strains are shown. The percentage of cells in a population containing Mmm1-GFP puncta is indicated. Bar: 5 µm.
Figure 6
Figure 6
GEM1 is not required for ERMES complex formation. Mmm1-GFP (and associated proteins) was immunopurified and the integrity of the ERMES complex analyzed by immunoblotting with antibodies against GFP or the endogenous proteins Mdm10 and Mdm12.
Figure 7
Figure 7
The ERMES complex is a stable structure. Dotted lines mark a cluster of three WT cells expressing the ERMES complex marker Mmm1-GFP imaged at 20 min intervals. Arrow marks a stable ERMES puncta; closed arrowhead marks a puncta moving into the growing bud; open circle marks a puncta that appears to form de novo. Bar: 5 µm.
Figure 8
Figure 8
Mmm1-GFP exchanges into a stable ERMES puncta. FRAP was performed on a WT strain expressing Mmm1-GFP to mark ERMES complexes. Time-lapse images of the ERMES puncta boxed in (A) before and after photobleaching at the indicated time points. The arrow marks the position of the photobleached puncta. Bar, 5 µm.
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