The network of prohibitin-interacting genes unraveled by our SGA analysis demonstrates an intimate functional relationship of prohibitins to the lipid composition of mitochondrial membranes (). While they are dispensable for yeast cell growth under normal conditions, prohibitins are essential for the integrity of the inner membrane and cell survival if membranes are deficient for CL or PE. Both CL and PE have similar physical properties and cluster into nonbilayer, hexagonal phase structures in lipid membranes (de Kruijff, 1997
). Disturbances in both biosynthetic pathways are synthetic lethal in yeast (Gohil et al., 2005
) and bacteria, illustrating that the physical similarities of CL and PE are of functional relevance in vivo. Consistently, clusters of PE and CL have been detected in bacterial membranes (Matsumoto et al., 2006
). It is therefore conceivable that defined lipid clusters with specific functions exist in the inner membrane of mitochondria. Contact sites between inner and outer mitochondrial membrane, at which import of nuclear-encoded mitochondrial proteins occurs (Reichert and Neupert, 2002
) and which have been linked to phospholipid transport processes (Simbeni et al., 1990
), were found to be enriched in PE and CL, and may represent such specialized membrane domains. Ringlike prohibitin complexes may serve as membrane organizers and affect the distribution of CL and PE in the membrane bilayer (). If CL and PE are present only at low concentrations, this function may become essential for inner membrane integrity and membrane-associated processes. Such an activity of prohibitins is in perfect accordance with the predicted function of prohibitins as protein scaffolds, and may ensure the recruitment of membrane proteins to a specific lipid environment. This includes m
-AAA proteases that assemble with prohibitins into large supercomplexes in the inner membrane (Steglich et al., 1999
). It should be noted that a fencelike activity of prohibitin ring complexes could also ensure the formation of protein-free domains in the mitochondrial inner membrane, which is considered to be the most protein-rich cellular membrane. These proposed functions of prohibitins as membrane organizers are reminiscent of other SPFH proteins like flotillins/reggies, which are distantly related to prohibitins and were found to induce microdomains in the plasma membrane and to modulate the assembly of signaling complexes (Frick et al., 2007
; Langhorst et al., 2008
Figure 8. Prohibitins and mitochondrial inner membrane organization. (A) Genetic interaction of prohibitins with PE and CL biosynthetic pathways. Synthetic lethal interactions between CHO1 and PGS1 and between PSD1 and CRD1 have been described previously (Janitor (more ...)
Altered levels of CL or PE compromise mitochondrial activities and are associated with many pathophysiological states, but the mechanisms that determine the phospholipid composition of mitochondrial membranes are poorly understood. Our findings identify the conserved family of Gep1-like proteins as novel membrane-associated regulators of CL and PE in mitochondrial membranes. Gep1 is essential for the accumulation of PE, whereas Ups1 is required for the accumulation of CL in mitochondrial membranes. Strikingly, deletion of GEP1
cells restored CL levels in mitochondria, which suggests competition between Gep1 and Ups1. Consistently, overexpression of Gep1 reduces CL in mitochondria. Thus, the phospholipid content of mitochondrial membranes critically depends on the level of Gep1. The competitive action of Gep1 and Ups1 allows the adjustment of relative PE and CL levels simply by modulating the amounts or the availability of the regulatory proteins. These findings demonstrate that mitochondrial levels of PE and CL are regulated coordinately by related proteins and are therefore in agreement with previous notions that cells may require a critical amount of these nonbilayer-forming phospholipids (Zhong et al., 2004
; Gohil et al., 2005
Gep1-like proteins likely exert a regulatory role during membrane biogenesis, as the mitochondrial phospholipid profile is only modestly altered in cells lacking all Gep1-like proteins. The decrease of the PE content of mitochondrial membranes in the absence of Gep1 is not caused by an impaired PE synthesis. Rather, PE does not accumulate stably in Gep1-deficient mitochondria, suggesting that Gep1 inhibits either a PE-specific lipase or the export of PE from mitochondria. Accordingly, the competition of Gep1 and Ups1 may determine the specificity of mitochondrial phospholipases or lipid transport processes. In agreement with an inhibitory role of Gep1 for PE export from mitochondria, we observed in the absence of Gep1 a slight increase of PC, which is generated by methyl transferases in ER membranes using mitochondrial PE (Kodaki and Yamashita, 1989
). Contact sites between inner and outer mitochondrial membranes have been discussed as sites of phospholipid transport (Simbeni et al., 1990
). It will be therefore of interest to examine whether Gep1-like proteins locate to these sites.
We identify the morphogenesis of cristae as one process critically dependent on mitochondrial PE. Decreased PE levels in Gep1- or Psd1-deficient cells compromise the processing of the dynamin-like GTPase Mgm1, which is required for membrane fusion and cristae formation (Meeusen et al., 2006
). Under these conditions, prohibitins are essential to maintain inner membrane integrity. Thus, residual Mgm1 processing sufficient to maintain mitochondrial cristae at decreased PE levels appears to critically depend on prohibitins. Notably, we have recently identified the processing of the mammalian Mgm1 homologue OPA1 as the central process controlled by prohibitins in mouse fibroblasts (Merkwirth et al., 2008
), which indicates that the same processes depend on prohibitin function in evolutionary distant organisms. Therefore, phenotypic differences associated with the loss of prohibitins in yeast and mammals likely reflect differences in the phospholipid profile of mitochondrial membranes or the lipid dependence of Mgm1/OPA1 processing itself.
Although the absence of CL did not inhibit Mgm1 processing in our experiments, we do not exclude a role of CL for proteolytic cleavage under certain growth conditions in yeast or in other organisms. Variations of the PE content of the inner membrane may mask the dependence of Mgm1 processing on CL. Accordingly, differences in the relative content of PE and CL may explain why the loss of Ups1 was observed previously to inhibit Mgm1 processing (Sesaki et al., 2006
). It is therefore an intriguing possibility that impaired processing of the mammalian Mgm1 homologue OPA1 causes the disturbed formation of mitochondrial cristae, which was observed in lymphoblasts of Barth syndrome patients or yeast cells lacking the CL transacylase tafazzin (Acehan et al., 2007
; Claypool et al., 2008
). The identification of Gep1-like proteins as regulators of CL and PE now allows for the direct examination of the role of an altered phospholipid composition for mitochondrial dysfunction in disease.
Gep1-like proteins and prohibitins acting as membrane organizers may affect various membrane-associated processes, which are known to depend on nonbilayer phospholipids. Mitochondrial fusion requires phospholipase D, which hydrolyzes CL in the outer membrane and generates the fusogenic lipid phosphatidic acid (Choi et al., 2006
). CL and PE affect also the insertion and oligomerization of the proapoptotic Bcl2-family member Bax in the outer membrane (Lucken-Ardjomande et al., 2008
). A CL deficiency in the inner membrane impairs the activity of mitochondrial enzymes (Jiang et al., 2000
), decreases the stability of respiratory chain supercomplexes and of mitochondrial DNA (Pfeiffer et al., 2003
), and accelerates apoptosis by facilitating the release of cytochrome c
from the intramitochondrial storage compartments (Choi et al., 2007
). It is conceivable that the assembly of the F1
ATP-synthase and respiratory complexes is yet another process dependent on defined functional domains within mitochondrial membranes. This is suggested by the synthetic interaction of prohibitins with genes coding for assembly factors of inner membrane complexes (Osman et al., 2007
), which do not drastically affect mitochondrial PE or CL levels when deleted.
Interestingly, mutations in the majority of GEP genes, which show a synthetic interaction with prohibitins, cause decreased levels of nonbilayer lipids. This includes MDM10
, which were originally identified as genes required for mitochondrial inheritance and DNA stability (Boldogh et al., 1998
) but were found more recently to function in the assembly of β-barrel proteins in the outer membrane (Meisinger et al., 2007
). Accordingly, an impaired insertion of an outer membrane protein affecting mitochondrial lipid biosynthesis may explain the reduced levels of PE and CL in the absence of Mdm10 or Mmm1. However, it cannot be excluded that both proteins play a direct role for the mitochondrial lipid metabolism and that an altered lipid composition causes deficiencies in the assembly of outer membrane proteins. Similarly, it is an attractive possibility that at least some of the GEP genes identified in this study, including previously uncharacterized open reading frames, regulate directly the biosynthesis of PE and CL in mitochondria and affect the activity of biosynthetic enzymes or intramitochondrial lipid transport processes. The identification of components mediating lipid import from the ER, transbilayer flipping across mitochondrial membranes, or lipid transport across the intermembrane space of mitochondria has yet be accomplished.