TA proteins form a unique group of membrane proteins that completely rely on posttranslational import for their insertion into either mitochondrial or ER membranes. This is a result of their single TMD existing at the most C-terminal region, the TA leaving the ribosome only upon completion of translation (Borgese et al., 2003b
). Recently, the cellular machinery that inserts most of the ER-bound TA proteins has been discovered in both yeast and mammals (Stefanovic and Hegde, 2007
; Schuldiner et al., 2008
; Jonikas et al., 2009
; Favaloro et al., 2010
). However, dedicated machinery for the biogenesis of mitochondrial TA proteins has not been identified.
By systematically screening for genes that take part in the biogenesis of the MOM TA protein Gem1, we could not find a single deletion strain that displayed accumulation of the protein in a cytosolic form or in which the protein could not be detected. These would be the phenotypes expected from mutants of dedicated insertion machinery, in which a preinserted form either accumulates in the cytosol or is rapidly degraded. Genetic screens have their limitations and, at least hypothetically, our results do not necessarily imply that no such factors exist. Our inability to find such a factor could potentially stem from a redundancy of the protein required for insertion (a single mutation would not demonstrate a phenotype robust enough to be detected). Another theoretical explanation is that if such machinery is essential (although none of the mitochondrial TA proteins is an essential one, and the dedicated ER machinery is also not essential), then the hypomorphic alleles of essential genes used in the screen might not have been compromised enough to induce a detectable phenotype. Finally, the mutant might not have been represented in our library, or we might not have screened under conditions that require such protein machinery (for example, GET complex–dependent insertion of ER TA proteins seems to be required only following the diauxic shift; Schuldiner et al., 2008
). To rule out these concerns, increase our coverage, and verify our results, we performed our screen twice with two different types of reporter proteins (either the TMD of Gem1 alone or the entire protein). Moreover, to minimize loss of strains due to the handling history of the library, we carried out the two screens with yeast deletion libraries taken from different sources. Finally, the screens were performed under two very different growth conditions, either on a semifermentable carbon source (galactose) in non–logarithmic growth phase or on a fully fermentable carbon source (glucose) during mid–logarithmic growth. Taking into consideration our extensive screens and the fact that previous work in vitro did not identify a proteinaceous component essential for TA protein insertion into the MOM (Setoguchi et al., 2006
; Kemper et al., 2008
), we suggest that MOM TA protein insertion in yeast most probably does not require dedicated proteins. It may be that in higher eukaryotes, in which many more mitochondrial TA proteins exist (Kalbfleisch et al., 2007
), a dedicated system might have evolved, but it has eluded identification to date (Setoguchi et al., 2006
Our screens did uncover a single protein, Spf1, that affects insertion of MOM TA proteins that are not subunits of the TOM complex, namely Gem1 and Fis1. It is currently unclear whether Spf1 plays a similarly important role in the biogenesis of Tom5, Tom6, and Tom7. The latter proteins may follow a different import pathway as several MOM proteins such as Mim1, Mas37/Sam37 and the Tom receptors were reported to be involved in their biogenesis (Dietmeier et al., 1997
; Stojanovski et al., 2007
; Thornton et al., 2010
). However, cofactors for correct biogenesis do not exclude a universal mechanism for the membrane insertion process. It could be that the small TOM components simply require additional proteins for downstream steps, such as assembly into the TOM complex. Although triple deletion in Neurospora crassa
, and TOM7
and double deletion in yeast of TOM5
and additional TOM components were reported to be lethal (Dietmeier et al., 1997
; Sherman et al., 2005
), the lack of obvious growth and morphology phenotypes in spf1
cells does not contradict a potential requirement for functional Spf1 in the biogenesis of these proteins. Under logarithmic growth conditions, spf1
mitochondria still display about half the normal Fis1 levels. Hence, these cells most likely still harbor adequate amounts of any TA protein that is affected in this background. It is anticipated that even reduced mitochondrial amounts of small TOM proteins are sufficient to maintain normal function of the TOM complex, which in turn can support normal growth and mitochondrial morphology.
Spf1 is the first yeast protein ever shown to affect insertion of MOM TA proteins. However, since the exact biochemical role of Spf1 is not yet known, it is currently impossible to determine the precise mechanism by which its deletion affects insertion. The fact that Spf1 resides in ER membranes suggests that its effect is indirect. Because spf1
cells display loss of differential sterol levels between MOM and ER membranes, and reduced ergosterol levels caused mistargeting in vivo, our findings strongly support the notion that Spf1, either directly or indirectly, regulates ergosterol levels and this in turn affects MOM TA protein insertion.
It is well documented that the ergosterol content of cellular membranes is tightly regulated, and even minor changes can result in major effects on cellular processes (Stuven et al., 2003
). Ergosterol levels in the ER are usually higher than those in mitochondria, and it was shown that ergosterol increases the rigidity of a membrane. The ER membrane should therefore consistently be an unfavorable target for spontaneous insertion of MOM TA proteins. In support of the role of sterols in insertion of TA proteins into membranes, it has been shown that insertion of MOM TA proteins into liposomes is inhibited by increased levels of sterols in the membrane (Kemper et al., 2008
). This is also the case for mitochondrial signal-anchored proteins, such as OM45 (Merklinger et al., 2012
). Conversely, when ER-derived microsomes were depleted of sterols, they became more rapidly targeted by the ER TA protein cytB5 in a manner that did not require protein factors nor energy (Brambillasca et al., 2005
). Taken together, the inability to identify a proteinaceous component as being required for MOM TA protein insertion and the idea that sterol levels have a major role in determining insertion specificity strongly suggest that the lipid composition of a membrane can be the determinant guiding MOM TA protein distribution in yeast.
The hypothesis that sterol levels themselves are responsible for insertion specificity raises the question of why none of the mutants in the biosynthetic pathway of ergosterol came up in our screen as affecting MOM TA proteins insertion. One possible answer is that the majority of proteins in this pathway are essential, and it may be that their DAmP alleles did not cause a reduction in sterol levels considerable enough to cause mislocalization that could pass our threshold of detection. In support of this assumption, only when we visualized Fis1 insertion on a highly compromised ERG9
mutant (Babiskin and Smolke, 2011
) or Gem1 insertion on a strongly repressed ERG10
strain did we find that they phenocopied the mislocalization observed in spf1
The idea that sterol levels have such a strong regulatory role in protein insertion raises new questions of how TA protein insertion efficiency, membrane specificity, and regulation of targeting can be achieved. One way to achieve both efficiency and specificity would be to target the mRNA encoding TA proteins before translation to mitochondrial membranes. Indeed, it has been shown that the mRNA of mitochondrial proteins is often localized to the surface of mitochondria (Marc et al., 2002
; Haim et al., 2007
; Saint-Georges et al., 2008
; Eliyahu et al., 2010
). Another pathway to achieve specificity would be selective degradation of proteins inserted into the wrong target membranes; however, there is no evidence in support of this. Finally, regulation of insertion could simply be dependent on the membrane composition of different organelles, which would imply that a regulatory network of proteins exists to maintain accurate levels of sterols in each membrane. Future investigations can address the mechanism of such a network.
Organization of eukaryotic cells into intracellular organelles allows the creation of distinct biochemical environments. By controlling the characteristics (e.g., fluidity, thickness, and charge) of their membranes, cells can create unique environments into which proteins can insert or bind. Indeed, understanding how the composition of boundary membranes controls the shape, size, and activity of the respective organelles is still a major question in cell biology. Our work demonstrates an important aspect of how organelle identity and function can be affected by changes in the lipid composition of its membranes.