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Mol Biol Cell. Nov 2005; 16(11): 5410–5417.
PMCID: PMC1266436
Role of Essential Genes in Mitochondrial Morphogenesis in Saccharomyces cerevisiaeD in Box
Katrin Altmann* and Benedikt Westermann*
* Institut für Zellbiologie
Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, 95440 Bayreuth, Germany
Benjamin Glick, Monitoring Editor
Address correspondence to: Benedikt Westermann (benedikt.westermann/at/uni-bayreuth.de).
Received July 26, 2005; Revised August 23, 2005; Accepted August 24, 2005.
Mitochondria are essential organelles of eukaryotic cells. Inheritance and maintenance of mitochondrial structure depend on cytoskeleton-mediated organelle transport and continuous membrane fusion and fission events. However, in Saccharomyces cerevisiae most of the known components involved in these processes are encoded by genes that are not essential for viability. Here we asked which essential genes are required for mitochondrial distribution and morphology. To address this question, we performed a systematic screen of a yeast strain collection harboring essential genes under control of a regulatable promoter. This library contains 768 yeast mutants and covers approximately two thirds of all essential yeast genes. A total of 119 essential genes were found to be required for maintenance of mitochondrial morphology. Among these, genes were highly enriched that encode proteins involved in ergosterol biosynthesis, mitochondrial protein import, actin-dependent transport processes, vesicular trafficking, and ubiquitin/26S proteasome-dependent protein degradation. We conclude that these cellular pathways play an important role in mitochondrial morphogenesis and inheritance.
Mitochondria supply the cell with ATP generated by oxidative phosphorylation (Saraste, 1999 blue right-pointing triangle), they are involved in the biosynthesis of many cellular metabolites (Scheffler, 2000 blue right-pointing triangle), they play a key role in the assembly of iron/sulfur clusters (Lill and Mühlenhoff, 2005 blue right-pointing triangle), and they are central regulators of programmed cell death (Desagher and Martinou, 2000 blue right-pointing triangle). Budding yeast Saccharomyces cerevisiae is an excellent model organism to study mitochondrial biogenesis and function. Because S. cerevisiae is a facultative anaerobic yeast capable of satisfying its energy requirements with ATP generated by fermentation, only few mitochondrial proteins are essential for viability in this organism. Among these are several factors required for biogenesis of iron/sulfur clusters that serve as cofactors for a number of mitochondrial and extramitochondrial enzymes (Lill and Mühlenhoff, 2005 blue right-pointing triangle). Other essential mitochondrial proteins are subunits of the molecular machines that mediate import, folding, and assembly of nuclear-encoded mitochondrial proteins (Neupert and Brunner, 2002 blue right-pointing triangle; Pfanner et al., 2004 blue right-pointing triangle; Rehling et al., 2004 blue right-pointing triangle). Recently, it was reported that a soluble ATP-binding cassette protein, Rli1, carries iron/sulfur clusters. Rli1 is a factor required for nuclear export of newly assembled subunits of cytosolic ribosomes and thus is essential for cell viability. As the biogenesis of iron/sulfur clusters requires the mitochondrial assembly machinery, the dependence of Rli1 on iron/sulfur clusters is sufficient to explain the essential nature of mitochondria in yeast (Kispal et al., 2005 blue right-pointing triangle; Yarunin et al., 2005 blue right-pointing triangle).
Because mitochondria cannot be generated de novo, they must be inherited during cell division (Warren and Wickner, 1996 blue right-pointing triangle). Inheritance of the organelles is mediated by cytoskeleton-mediated transport, and maintenance of mitochondrial structure and function depends on continuous fusion and fission events (Bereiter-Hahn and Vöth, 1994 blue right-pointing triangle; Yaffe, 1999 blue right-pointing triangle). In recent years, an increasing number of proteins essential for mitochondrial inheritance and structure have been identified in yeast. These include factors of the mitochondrial membrane fusion and division machineries as well as proteins involved in structural maintenance of the organelle and cytoskeleton-dependent motility (Hermann and Shaw, 1998 blue right-pointing triangle; Jensen et al., 2000 blue right-pointing triangle; Boldogh et al., 2001b blue right-pointing triangle; Dimmer et al., 2002 blue right-pointing triangle; Shaw and Nunnari, 2002 blue right-pointing triangle; Scott et al., 2003 blue right-pointing triangle). Surprisingly, all components specifically required for mitochondrial dynamics are encoded by nonessential genes. However, some synthetic lethal relationships have been revealed. Deletion of the genes encoding the mitochondrial inheritance components Mdm10, Mdm12, and Mmm1 is synthetically lethal with deletion of the genes encoding prohibitin family members (Berger and Yaffe, 1998 blue right-pointing triangle); and deletion of the MDM10, MDM12, MMM1, and MMM2 genes is synthetically lethal with deletion of the MDM31 and MDM32 genes (Dimmer et al., 2005 blue right-pointing triangle). Furthermore, overexpression of Mdm33, an inner membrane protein required for mitochondrial morphogenesis, is lethal (Messerschmitt et al., 2003 blue right-pointing triangle). These genetic findings underscore the fact that inheritance of mitochondria is an essential process.
To identify essential genes required for mitochondrial distribution and morphology in yeast, we screened a library containing mutants harboring essential genes that have been placed under control of a regulatable promoter (Mnaimneh et al., 2004 blue right-pointing triangle). Assignment of mutants with aberrant mitochondrial morphology to functional classes identified the essential cellular pathways that are important for mitochondrial morphogenesis.
Yeast Genetic Methods
S. cerevisiae was cultivated and manipulated according to standard procedures (Sherman, 1991 blue right-pointing triangle). Yeast Tet regulated ORF clone collection and update, the strain collections used for expression of essential genes from a regulatable promoter (Gari et al., 1997 blue right-pointing triangle; Mnaimneh et al., 2004 blue right-pointing triangle), were obtained from BioCat (Heidelberg, Germany). Repression of the TetO7 promoter was achieved by the addition of 10 μg/ml doxycycline to the medium and incubation over night or for up to 3 d.
Screening for Essential Genes Involved in Mitochondrial Morphogenesis
After thawing 96-well plates containing frozen stocks of the yeast strains, cells were transferred to yeast extract/peptone/glucose (YPD) plates without or with 10 μg/ml doxycycline using a sterile pinning tool and incubated at 30°C over night. Media lacking doxycycline allowed expression of essential genes from the TetO7 promoter, and these cultures served as negative controls. Media containing 10 μg/ml doxycycline (YPD/Dox) efficiently repress the TetO7 promoter (Gari et al., 1997 blue right-pointing triangle; Mnaimneh et al., 2004 blue right-pointing triangle). About one third of the strains failed to grow on the YPD/Dox plate. Strains that did grow were subjected to two additional rounds of replica plating on fresh YPD/Dox plates using a pinning tool. About half of the strains were still able to grow on the third YPD/Dox plate. For these strains, growth could be observed even after seven rounds of replica plating.
For screening for mitochondrial morphology mutants, strains were inoculated in 0.5 ml YPD or YPD/Dox and grown over night at 30°C. YPD cultures were inoculated from YPD plates. YPD/Dox cultures were inoculated from YPD plates for strains that failed to grow on the first YPD/Dox plate, or from the first YPD/Dox plate for strains that failed to grow on the second YPD/Dox plate, or from the second YPD/Dox plate for strains that failed to grow on the third YPD/Dox plate, or from the third YPD/Dox plate for all remaining strains. The next morning, 3 ml fresh medium was added, and incubation was continued for at least 3 h. Mitochondria were stained by the addition of 0.1 μM rhodamine B hexyl ester (Molecular Probes, Eugene, OR) and inspected by fluorescence microscopy. Strains (n = 183) that showed aberrant mitochondrial morphology on YPD/Dox media, or that did not stain well, were transformed with pYX142-mtGFP expressing mitochondria-targeted GFP (Westermann and Neupert, 2000 blue right-pointing triangle). These transformants were subjected to another round of screening to identify clones that reproducibly do not exhibit wild-type-like mitochondria under promoter shut off conditions. All rounds of screening were performed without prior reference to strain identity. To exclude the possibility that mitochondrial phenotypes were indirect consequences of cell death in the presence of doxycycline, cultures were stained with FUN 1 (LIVE/DEAD yeast viability kit, Molecular Probes) to score for viability of cells (Millard et al., 1997 blue right-pointing triangle). With very few exceptions (as indicated in Supplementary Table 1) 70–100% of the cells grown in YPD/Dox media were alive and metabolically active under screening conditions.
Staining of Cellular Structures and Microscopy
Mitochondria were stained in living cells with 0.1 μM rhodamine B hexyl ester (Molecular Probes) or mitochondria-targeted GFP expressed from pYX142-mtGFP (Westermann and Neupert, 2000 blue right-pointing triangle; Dimmer et al., 2002 blue right-pointing triangle). The actin cytoskeleton was stained with rhodamine-phalloidin (Molecular Probes) as described (Amberg, 1998 blue right-pointing triangle). Differential interference contrast (DIC) and epifluorescence microscopy was performed using an Axioplan 2 microscope equipped with a Plan-Neofluar 100×/1.30 Ph3 oil objective (Carl Zeiss Jena GmbH, Oberkochen, Germany). Images were recorded with an Evolution VF Mono Cooled monochrome camera (Intas, Göttingen, Germany) and processed with Image ProPlus 5.0 and ScopePro 4.5 software (Media Cybernetics, Silver Spring, MD).
Identification of Essential Cellular Functions Required for Mitochondrial Morphogenesis
To identify essential genes required for establishment and maintenance of normal mitochondrial distribution and morphology, we systematically screened a promoter shut off yeast strain collection that covers approximately two thirds of all essential yeast genes (Mnaimneh et al., 2004 blue right-pointing triangle). The 768 strains contained in this collection were incubated under permissive and restrictive conditions, stained with mitochondria-specific probes and screened for mutants that showed aberrant mitochondrial morphology upon depletion of essential gene products. A total number of 119 mutants (corresponding to 15% of the strains present in the collection) were identified that reproducibly showed severe defects in the organization of their mitochondria (see Supplementary Table 1 for a complete list).
Based on information obtained from the Saccharomyces Genome Database (Christie et al., 2004 blue right-pointing triangle) and the Comprehensive Yeast Genome Database (Güldener et al., 2005 blue right-pointing triangle) and manual annotation, the strains present in the collection were grouped into functional categories (see Supplementary Table 1). The following five functional categories were identified that contained a high proportion of mutants with aberrant mitochondria: (1) ergosterol biosynthesis, with 100% of the screened mutants being affected; (2) mitochondrial protein import, 70% affected; (3) actin cytoskeleton-associated proteins, 67% affected; (4) vesicular transport and secretion, 61% affected; and (5) ubiquitin/26S proteasome-dependent protein degradation, 32% affected. In contrast, only 7% of the mutants lacking essential proteins of other functions showed aberrant mitochondria. A similar number, 8%, was obtained for mutants lacking proteins of unknown function. These results are summarized in Figure 1, Table 1, and Supplementary Table 1. We conclude that mitochondrial morphogenesis and inheritance rely on the presence of a complete complement of ergosterol biosynthetic enzymes and intact machineries of mitochondrial protein import, actin cytoskeleton-dependent motility, vesicular transport, and proteasome-dependent protein degradation.
Figure 1.
Figure 1.
Essential cellular processes required for maintenance of mitochondrial morphology. Promoter shutoff strains (n = 768) lacking essential gene products were screened for mutants with aberrant mitochondrial morphology. Of these strains, 15% showed strong (more ...)
Table 1.
Table 1.
Essential cellular pathways, protein complexes, and proteins required for mitochondrial morphogenesis in yeast
Role of Ergosterol Biosynthesis in Maintenance of Mitochondrial Morphology
Mutants lacking enzymes required for the biosynthesis of ergosterol showed clumped and swollen mitochondria (Table 1 and Supplementary Table 1). This is exemplified for erg7 and erg8 (Figure 2), whereas the other mutants looked very similar. When the strains were grown in the absence of doxycycline, i.e., under permissive conditions, they exhibited the characteristic mitochondrial tubular network of wild-type cells (Figure 2). Addition of doxycycline to the growth medium did not affect mitochondrial morphology of wild-type cells, indicating that the observed defects were consequences of depletion of essential gene products (Figure 2).
Figure 2.
Figure 2.
Yeast cells lacking ergosterol biosynthetic enzymes harbor aberrant mitochondria. Strains expressing mitochondria-targeted GFP were grown in the absence (- Dox) or presence (+ Dox) of doxycycline in YPD medium to logarithmic growth phase and analyzed (more ...)
The fact that all strains with down-regulated ergosterol biosynthetic enzymes showed severe mitochondrial morphology defects points to an essential role of this membrane lipid for maintenance of the structure of membrane-bounded organelles. Interestingly, ergosterol has been reported to be required for the priming step of homotypic vacuole fusion in yeast (Kato and Wickner, 2001 blue right-pointing triangle). Because vacuolar membranes have relatively low ergosterol content, the role of this lipid in membrane fusion presumably is not the modulation of the physical properties of the membrane. Rather, it has been suggested to specifically activate and/or rearrange membrane proteins required for the vacuolar fusion reaction (Kato and Wickner, 2001 blue right-pointing triangle; Fratti et al., 2004 blue right-pointing triangle). The ergosterol content of mitochondrial membranes (6 μg ergosterol per mg of organellar protein in the outer membrane and 25 μg/mg in the inner membrane) is even lower than that of vacuolar membranes (49 μg/mg; Zinser et al., 1993 blue right-pointing triangle). Thus, it is conceivable that also in the case of mitochondria, ergosterol may have a modulatory function on the machinery of membrane fusion and/or fission.
Link between the Mitochondrial Protein Import/Assembly Machinery and Mitochondrial Morphology
Many mutants lacking proteins involved in mitochondrial protein translocation, sorting, and assembly showed severe defects in mitochondrial morphology. These include essential subunits of the TOM complex (translocase of the outer membrane), the SAM complex (sorting and assembly machinery of the outer membrane), and the TIM23 complex (translocase of the inner membrane).1 The following proteins were found to be required for normal mitochondrial morphology: Tom22, a subunit of the TOM complex; Mim1, a protein required for TOM complex assembly; Sam35 and Sam50, two subunits of the SAM complex; and Pam18, Zim17, and Mge1, three components or cofactors of the TIM23 translocase and its associated import motor (Figure 3 and Table 1). Strains that showed normal mitochondria in the presence of doxycycline include mas1, lacking a subunit of the mitochondrial processing peptidase; tim50, lacking a subunit of the TIM23 translocase; and tim22, lacking a subunit of the TIM22 translocase. It is presently unclear whether residual amounts of these proteins are sufficient to maintain normal mitochondrial shape or whether these proteins are not required for mitochondrial morphogenesis.
Figure 3.
Figure 3.
Yeast cells lacking components of the mitochondrial protein import and sorting machinery harbor aberrant mitochondria. Yeast strains were analyzed as in Figure 2. Bar, 5 μm.
Because 70% of the strains with down-regulated components of the mitochondrial protein sorting machinery showed aberrant mitochondria, we conclude that intact TOM, SAM, and TIM23 complexes are required for mitochondrial morphogenesis. This is in accordance with our earlier observation that a nonessential TOM component, Tom7, is required for wild-type-like mitochondrial distribution and morphology (Dimmer et al., 2002 blue right-pointing triangle). In case of Sam50, similar mitochondrial defects have been reported in a recent study using temperature-sensitive sam50 mutants. It has been suggested that Sam50 together with Mas37 and the mitochondrial morphology protein Mdm10 is required for assembly of Tom40 and its association with Tom22 to generate a functional TOM complex (Meisinger et al., 2004 blue right-pointing triangle). Most of the known proteins required for maintenance of mitochondrial structure are located in the mitochondrial outer and inner membrane. Because these proteins have to be imported via the TOM complex and then sorted to the mitochondrial subcompartments, it is conceivable that defects of the mitochondrial protein sorting machinery lead to aberrant mitochondrial morphology as a secondary consequence.
Dependence of Mitochondrial Morphology on the Actin Cytoskeleton
Mitochondrial transport in S. cerevisiae is dependent on actin filaments (Hermann and Shaw, 1998 blue right-pointing triangle; Jensen et al., 2000 blue right-pointing triangle; Boldogh et al., 2001b blue right-pointing triangle). Therefore, we expected that depletion of actin cytoskeleton-related factors would result in mitochondrial morphology defects. Mutants of 15 essential yeast genes required for integrity or function of the actin cytoskeleton are contained in the strain collection. We examined the organization of mitochondria and the actin cytoskeleton in all of these mutants by fluorescence microscopy. Based on their phenotypes, the mutants were grouped into four classes: class A mutants have aberrant mitochondria and disorganized actin filaments; class B mutants have aberrant mitochondria and normal actin filaments; class C mutants have normal mitochondria and disorganized actin filaments; and class D mutants have normal mitochondria and normal actin filaments (Figure 4). Class A comprises subunits of the ARP2/3 complex (Arc35, Arc40, Arp2), subunits of the CCT chaperone, which is required for assembly of actin and tubulin (Cct4, Cct6), a myosin-related motor protein (Myo2), and other factors required for organization of the actin cytoskeleton (Cof1/cofilin, Iqg1, Pfy1/profilin). Class B contains Mlc1, a myosin light chain associated with Myo2. Class C comprises a myosin-related motor protein (Myo1), and a component of cortical actin patches (Las17). Class D comprises three proteins involved in actin cytoskeleton assembly (Mss4, Pan1, Sda1). Class C and D components apparently do not play an important role in mitochondrial morphogenesis.
Figure 4
Figure 4
(facing page). Yeast cells lacking components linked to the actin cytoskeleton harbor aberrant mitochondria. Strains expressing mitochondria-targeted GFP were grown in the absence (- Dox) or presence (+ Dox) of doxycycline in YPD medium to logarithmic (more ...)
The large number of actin cytoskeleton mutants with aberrant mitochondria found in classes A and B underscores the fundamental importance of actin-dependent transport for mitochondrial distribution and morphology in yeast. Although the mitochondrial phenotype seen in cells lacking Cof1, Iqg1, Pfy1, and CCT subunits is most likely an indirect consequence of disturbed microfilament organization, a direct role in mitochondrial binding to actin has been suggested for the ARP2/3 complex (Boldogh et al., 2001a blue right-pointing triangle; Fehrenbacher et al., 2004 blue right-pointing triangle) and Myo2 (Itoh et al., 2002 blue right-pointing triangle; Boldogh et al., 2004 blue right-pointing triangle; Itoh et al., 2004 blue right-pointing triangle). It is still a matter of debate whether ARP2/3 complex-driven motility or myosin-related motor proteins are of major importance for mitochondrial movement (Boldogh et al., 2001a blue right-pointing triangle, 2004 blue right-pointing triangle; Itoh et al., 2004 blue right-pointing triangle). Here we found that the mlc1 mutant harbors intact actin cables, but highly aberrant mitochondria. Intriguingly, mitochondrial defects were also apparent in mlc1 and myo2 mutants in the absence of doxycycline (Figure 4), conditions that likely lead to nonphysiological expression levels of these proteins. The fact that mitochondrial defects are manifest in cells that display a relatively normal actin cytoskeleton is in favor of a direct role of Myo2 and its associated light chain, Mlc1, in linking the organelle to the cytoskeleton.
Connection of Vesicular Transport and Mitochondrial Morphology
Components of the vesicular trafficking system constitute the largest group of essential yeast proteins that were identified as mitochondrial biogenesis components. Thirty-five proteins mediating protein translocation into the ER and vesicle budding and fusion were found to be required for maintenance of mitochondrial morphology (Table 1 and Supplementary Table 1). On depletion of these proteins, mitochondria appeared fragmented and/or aggregated. This phenotype was very similar in all mutants. Examples shown in Figure 5 are Sec17 and Sec18, homologues of mammalian alpha-SNAP and NSF, which are required for priming of the vesicular fusion machinery, Sec63, a subunit of the SEC61 translocon, and Srp101, the alpha subunit of the signal recognition particle receptor.
Figure 5.
Figure 5.
Yeast cells lacking components of the protein secretion and vesicular trafficking machinery harbor aberrant mitochondria. Yeast strains were analyzed as in Figure 2. Bar, 5 μm.
Previous observations of mitochondria in mutants of the secretory pathway have yielded ambiguous results. For example, temperature-sensitive sec18 mutants did not show defects in mitochondrial fusion (Nunnari et al., 1997 blue right-pointing triangle), whereas temperature-sensitive srp101 mutants contained fragmented mitochondria (Prinz et al., 2000 blue right-pointing triangle), similar to the aberrant organelles observed here (Figure 5). It is thought that defects in mitochondrial structure do not affect the ER, because mutants such as fzo1-1, Δmdm30, Δmdm31, Δmdm32, Δmdm33, mmm1-1, Δmmm2, and Δugo1 maintain a normal ER structure (Prinz et al., 2000 blue right-pointing triangle; Sesaki and Jensen, 2001 blue right-pointing triangle; Fritz et al., 2003 blue right-pointing triangle; Messerschmitt et al., 2003 blue right-pointing triangle; Youngman et al., 2004 blue right-pointing triangle; Dimmer et al., 2005 blue right-pointing triangle). In contrast, the majority of vesicular transport mutants analyzed here showed strong defects in mitochondrial morphology. We conclude that the ER and secretory pathway play an important and general role in maintenance of a normal mitochondrial reticulum. We consider it likely that this role involves the supply of mitochondria with lipids.
Role of the Ubiquitin/26S Proteasome System for Mitochondrial Biogenesis
A large fraction, 32%, of the mutants lacking proteins of the ubiquitin/26S proteasome system showed strong defects in mitochondrial morphology. When only proteasome subunits are considered, this number increases to 64% (Figure 1), whereas strains lacking cell cycle-specific protein degradation factors, such as components of the anaphase-promoting complex, showed no mitochondrial defects (see Supplementary Table 1). In addition to seven proteasome subunits (Table 1) the following components were found to be required for normal mitochondrial structure: two subunits of the SCF ubiquitin ligase complex (Cdc34 and Cdc53/cullin), a ubiquitin-activating enzyme (Uba1), and a protein involved in recognition of ubiquitinated proteins (Ufd1). Mitochondria generally appeared highly fragmented or aggregated in these mutants, as shown for cdc34, cdc53, pre1, and rpn8 (Figure 6). These results are in accordance with earlier observations that ubiquitin (Fisk and Yaffe, 1999 blue right-pointing triangle), the 26S proteasome (Rinaldi et al., 1998 blue right-pointing triangle) and SCF-dependent protein degradation (Fritz et al., 2003 blue right-pointing triangle) are involved in maintenance of mitochondrial morphology.
Figure 6.
Figure 6.
Yeast cells lacking components of the ubiquitin/26S proteasome system harbor aberrant mitochondria. Yeast strains were analyzed as in Figure 2. Bar, 5 μm.
Proteins of Unknown Function
Four mutants lacking proteins of unknown function showed severe mitochondrial defects, ymr134, ydr339c, ynl149c (and its overlapping ORF ynl150w), and yor060c (Figure 7). These ORFs might encode novel essential proteins required for mitochondrial morphogenesis. However, the percentage of mutants showing aberrant mitochondria is very similar for ORFs of unknown function (8%) and for the group of all remaining mutants (7%; Supplementary Table 1). We consider it likely that the latter mutants show aberrant mitochondria due to pleiotropic effects of their mutations or general defects in cell physiology. It remains to be determined whether the novel components of unknown function might contribute to mitochondrial inheritance directly or indirectly.
Figure 7.
Figure 7.
Yeast cells lacking components of unknown function harbor aberrant mitochondria. Yeast strains were analyzed as in Figure 2. Bar, 5 μm.
With the screening of 4794 deletion mutants lacking nonessential genes (Dimmer et al., 2002 blue right-pointing triangle) and the screening of 768 promoter shutoff strains reported here we have now systematically analyzed 5562 of the ca. 6000 yeast genes for their role in mitochondrial distribution and morphology. Most of the known components mediating mitochondrial fusion, fission, and maintenance of mitochondrial structure are encoded by nonessential genes (Hermann and Shaw, 1998 blue right-pointing triangle; Jensen et al., 2000 blue right-pointing triangle; Boldogh et al., 2001b blue right-pointing triangle; Dimmer et al., 2002 blue right-pointing triangle; Shaw and Nunnari, 2002 blue right-pointing triangle; Scott et al., 2003 blue right-pointing triangle). The present work allows us to define the essential cellular pathways that contribute to the complex process of mitochondrial inheritance. In particular, our results have revealed an important role of ergosterol biosynthesis, mitochondrial protein import, and vesicular trafficking. Apparently, these processes are much more important for mitochondrial morphogenesis than previously anticipated. Moreover, our results underscore the fundamental importance of the actin cytoskeleton and the proteasome for mitochondrial biogenesis.
Obviously, many of the identified components affect mitochondrial morphology in a rather indirect way, as, for example, subunits of the mitochondrial protein translocation machinery or the vesicular transport system. Moreover, depletion of essential gene products for several days may lead to pleiotropic defects, including aberrant mitochondrial morphology. It should be noted that such pleiotropic defects might play a role in all classes of proteins reported here. On the other hand, some of the components identified (e.g., Dpm1, Mot1, Ncp1, Prp31, Sec4, and Sec63) were recently found in the mitochondrial proteome (Sickmann et al., 2003 blue right-pointing triangle) although they were previously localized to other cellular compartments. This opens the exciting possibility that these proteins have a dual localization and might play a more direct role in mitochondrial biogenesis. Clearly, much more work is required to reveal the molecular interactions that link mitochondrial morphogenesis with cellular processes such as synthesis and exchange of membrane lipids, mitochondrial protein import, cytoskeleton-dependent organelle transport, and protein degradation.
Supplementary Material
[Supplementary Material]
Acknowledgments
We thank Jan Fiedler for his contributions to some experiments and the Deutsche Forschungsgemeinschaft for financial support through grants We 2174/2–4 and We 2174/3–1.
Notes
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-07-0678) on August 31, 2005.
Abbreviations used: Dox, doxycycline; YPD, yeast extract/peptone/glucose.
D in BoxThe online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Footnotes
1Many components of mitochondrial protein sorting machineries have alternative names. Here, we use the standard gene names according to the Saccharomyces Genome Database.
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