Suppression of a Defect in Mitochondrial Protein Import Identifies Cytosolic Proteins Required for Viability of Yeast Cells Lacking Mitochondrial DNA

Corresponding author: Robert E. Jensen, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205., rjensen{at}jhmi.edu (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The TIM22 complex, required for the insertion of imported polytopic proteins into the mitochondrial inner membrane, contains the nonessential Tim18p subunit. To learn more about the function of Tim18p, we screened for high-copy suppressors of the inability of tim18 mutants to live without mitochondrial DNA (mtDNA). We identified several genes encoding cytosolic proteins, including CCT6, SSB1, ICY1, TIP41, and PBP1, which, when overproduced, rescue the mtDNA dependence of tim18 cells. Furthermore, these same plasmids rescue the petite-negative phenotype of cells lacking other components of the mitochondrial protein import machinery. Strikingly, disruption of the genes identified by the different suppressors produces cells that are unable to grow without mtDNA. We speculate that loss of mtDNA leads to a lowered inner membrane potential, and subtle changes in import efficiency can no longer be tolerated. Our results suggest that increased amounts of Cct6p, Ssb1p, Icy1p, Tip41p, and Pbp1p help overcome the problems resulting from a defect in protein import.

WHILE mitochondria contain their own DNA (mtDNA) encoding a handful of proteins, the vast majority of mitochondrial proteins are synthesized on cytosolic ribosomes and imported post-translationally into the organelle (JENSEN and DUNN 2002 ; PFANNER and CHACINSKA 2002 ). These proteins are sorted to four possible locations within the mitochondria: the outer membrane (OM), the intermembrane space (IMS), the inner membrane (IM), and the matrix. Several multi-subunit transport machines in the OM and IM, called translocons, mediate protein import. Imported proteins first travel through a translocon in the OM, called the translocon of the outer membrane (TOM) complex. Many proteins are further transported through the IM by translocons of the inner membrane (TIM) complexes.

The TOM and TIM channels are best characterized in fungi such as Neurospora crassa and Saccharomyces cerevisae, although many of the identified components have homologs in higher eukaryotes (HOOGENRAAD et al. 2002 ). Precursors in the cytosol are recognized by one of several receptor proteins on the surface of mitochondria: Tom70p, Tom22p, and Tom20p. After binding to receptors, proteins cross the OM via the TOM translocon, which forms a pore in the mitochondrial OM. Mitochondrial precursors containing a cleavable presequence are then imported into the matrix by the action of the inner-membrane-localized TIM23 complex. The TIM23 complex consists of the Tim50, Tim23, and Tim17 proteins, which may form and/or regulate a protein-translocating pore (TRUSCOTT et al. 2001 ; GEISSLER et al. 2002 ; YAMAMOTO et al. 2002 ). In addition, Tim44p and mt-Hsp70 act as a molecular motor to ensure unidirectional movement of the precursor through the TIM23 channel (NEUPERT and BRUNNER 2002 ; VOOS and ROTTGERS 2002 ). Once a precursor reaches the matrix, its presequence is removed by a processing peptidase (GAKH et al. 2002 ).

Many proteins that end up in the mitochondria use a different TIM complex for their import. In particular, a subset of polytopic membrane proteins, or proteins that pass through the membrane several times, is inserted into the IM by the TIM22 translocon. The movement of these proteins from the TOM translocon to the TIM22 translocon is dependent upon soluble complexes in the IMS, such as the TIM9/10 or TIM8/13 complex (KOEHLER et al. 1999 ). Within the TIM22 complex, the Tim22 protein, which is homologous to both Tim23p and Tim17p (SIRRENBERG et al. 1996 ), is thought to form a pore through which polytopic proteins are inserted into the IM (KOVERMANN et al. 2002 ). Tim54p and Tim18p are membrane-spanning proteins that are also members of the TIM22 translocon, but their function in polytopic protein insertion is not yet known (KERSCHER et al. 1997 , KERSCHER et al. 2000 ; KOEHLER et al. 2000 ).

An electrochemical gradient across the IM is essential for the activity of both TIM complexes (SCHLEYER et al. 1982 ; ZWIZINSKI et al. 1983 ; EILERS et al. 1987 ; MARTIN et al. 1991 ). This potential is usually generated by the export of protons out of the matrix by the respiratory chain, components of which are encoded in the nucleus and by mitochondrial DNA. When the respiratory chain is damaged by mutation or when cells lose mtDNA, creating "petite" cells, the IM potential is thought to be formed by the activities of an ATP/ADP transporter and of the matrix-localized F₁ ATPase (DUPONT et al. 1985 ; GIRAUD and VELOURS 1997 ). The exchange of ATP^-4 for ADP^-3 in some way produces a sufficient potential for mitochondrial function. ATP/ADP transport is mediated by carrier proteins in the IM, with Aac2p being the most important (CHEN and CLARK-WALKER 2000 ).

The Tim18 protein is a nonessential member of the TIM22 complex, but tim18 cells cannot live without mtDNA and are cold sensitive when grown on rich glucose medium (KERSCHER et al. 2000 ). To further explore the function of the inner-membrane-localized Tim18 protein, we isolated high-copy suppressors of the tim18 mutant. Surprisingly, all of our suppressors encoded cytosolic proteins, including CCT6, SSB1, TIP41, ICY1, and PBP1. We present data indicating that a genetic pathway stretching from the cytosol to the mitochondrial matrix is required for viability of cells lacking mtDNA. We also speculate on how the proteins within this pathway might function when mitochondrial protein import is compromised.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and media:
The genotypes of strains used in this study are indicated in Table 1. RJ992 was generated as previously described (KERSCHER et al. 2000 ). tim18 strain RJ1263 was constructed by first mating RJ867 and RJ868, and then PCR-mediated gene disruption was used to delete one copy of TIM18 in the diploid. After sporulation and tetrad dissection, the tim18::HIS3 segregant RJ1263 was isolated. ⁰ strain RJ1582 was obtained by growing RJ868 cells twice to saturation in YEPD containing ethidium bromide (EtBr). RJ1582 was identified as a single colony that failed to grow on YEP medium containing glycerol and ethanol as the sole carbon source and was subsequently shown to contain no mtDNA by microscopy after staining with 1 µg/ml 4', 6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) for 15 min. The op1 strain RJ1581 was generated by crossing strain RJ1577 to strain RJ1578. Strains RJ1577, RJ1583, RJ1584, RJ1585, RJ1586, RJ1587, RJ1605, and RJ1606 were obtained from Research Genetics (Huntsville, AL).

Standard yeast genetic techniques and media were used (ADAMS et al. 1997 ), except that SD complete medium was made with individual amino acids and not casamino acids. Cycloheximide (Calbiochem, La Jolla, CA) and ethidium bromide (Sigma, St. Louis) were added to medium at 0.2 and 25 µg/ml, respectively. Sporulation was performed as follows: diploids were grown in GNA presporulation medium (5% dextrose, 3% Difco nutrient broth, 1% Difco yeast extract, 2% Difco bacto-agar; Saccharomyces Genome Database; http://genome-www.stanford.edu/Saccharomyces/) for three to five generations and then transferred to sporulation medium (0.01 g/ml potassium acetate, 2.5 x 10^-5 g/ml zinc acetate).

Isolation and identification of tim18 suppressors:
tim18 strain RJ992 was grown in YEPD to an OD₆₀₀ of 0.6 at 34° and then transformed with a 2µ-URA3 library containing genomic yeast DNA inserts of 6–8 kbp (a gift of Phillip Hieter, University of British Columbia). Ura⁺ transformants were selected for growth upon SD-Ura medium containing ethidium bromide (SD-Ura + EtBr) at 34°. Fifty-six large- and medium-sized colonies were picked, and the plasmid DNA was isolated (HOFFMAN and WINSTON 1987 ) and electroporated into bacterial cells. PCR analysis using oligonucleotides complementary to the TIM18 gene showed that 40 of the plasmids encoded Tim18p. One of the isolated plasmids carrying TIM18, called pM346, was used as a control in some of our studies. The remaining 16 plasmids were shown to suppress the tim18 mutation upon retransformation into yeast. Limited DNA sequencing of one or both ends of the genomic DNA (Biosynthesis and Sequencing Facility, Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore) allowed us to identify the chromosomal region contained within the DNA insert by searching Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/). Many of the plasmids contained the same or overlapping inserts, such that five different chromosomal regions were represented by the 16 plasmids.

Plasmid constructions:
To localize the genes responsible for suppression, subclones from the five different genomic DNA inserts were introduced into the 2µ-URA3 plasmid pRS426 (SIKORSKI and HIETER 1989 ) and transformed into tim18 strain RJ992. Ura⁺ transformants were streaked out onto SD-Ura plates containing 25 µg/ml EtBr to induce loss of mtDNA and allowed to grow at 34° for 3 days. Cells were then streaked onto SD-Ura medium lacking EtBr and the plates incubated at 34° for 4 days. If the plasmid suppressed the tim18 defect, then the cells would grow following the EtBr treatment; cells without functional suppressors would not grow.

The five plasmids that encode functional subclones were all constructed in similar ways. Genes were amplified from genomic yeast DNA using specific oligonucleotides and the polymerase chain reaction (PCR). The PCR product was digested with the appropriate restriction endonucleases and then inserted into the 2µ-URA3 vector pRS426 (SIKORSKI and HIETER 1989 ). The oligonucleotides and enzymes used for each subclone are as follows. pM350, which carries CCT6, was constructed using oligos 528 (5'-cagcctcgagacgagaacggtaagcatg-3') and 529 (5'-cggcctcgaggcaactggcaatgactat-3'), followed by XhoI digestion. pM351, which carries SSB1, was made with oligos 530 (5'-cgcggatccgagtacacacgggacttg-3') and 531 (5'-cgcggatccgcgttaccggcactgatt-3') and BamHI. For pM352, which carries ICY1, we used oligos 534 (5'-cgcggatccagctcgattttcagcacc-3') and 535 (5'-cgcggatccttccggtagctggtctta-3') and BamHI. pM353, which contains PBP1, was made using oligos 740 (5'-ccgctcgagatgagtcgcaccaagataag-3') and 741 (5'-ccgctcgagtggcgattgaaatactgattac-3') and XhoI. pM362, which carries TOM70, was constructed using oligos 705 (5'-ccgctcgagggatagggatggacaatagc-3') and 744 (5'-ataagaatgcggccgcgctccgcaaattggcgaggg-3') and both XhoI and NotI. pM354, which carries TOM70 in a URA3-CEN6 plasmid was made by inserting the XhoI/NotI TOM70-containing insert from pM362 into pRS316 (SIKORSKI and HIETER 1989 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cytosolic proteins can suppress the mtDNA dependence of the tim18 mutant:
As an attempt to determine the function of the Tim18 protein in mitochondrial protein import, we searched for genes that genetically interact with TIM18. Tim18p is not essential, but a strain deleted of TIM18 exhibits at least two phenotypes. First, cells lacking Tim18p are cold sensitive on rich medium containing glucose as the carbon source. Second, tim18 cells are inviable after plating onto medium containing EtBr (KERSCHER et al. 2000 ). This treatment causes the rapid loss of mtDNA (GOLDRING et al. 1970 ). Inability to grow after mtDNA loss is called a petite-negative phenotype (CHEN and CLARK-WALKER 2000 ). We used the petite-negative phenotype of the tim18 mutant as the basis for our high-copy suppression screen.

We transformed a tim18 strain with a yeast genomic library on a 2µ-URA3 vector. Plasmids containing the 2µ origin of replication are maintained at high copy, often resulting in the overexpression of genes carried on those plasmids (RINE 1991 ). We picked 56 colonies that grew on EtBr-containing medium and isolated the plasmid DNA from each strain. Plasmids from 40 colonies were found to contain the TIM18 gene and were discarded. Sixteen plasmids that did not encode Tim18p were subjected to limited sequencing of the genomic DNA inserts and found to represent five different chromosomal regions. Subclones containing different open reading frames from the inserts were then tested for the ability to suppress the tim18 mutant as described in MATERIALS AND METHODS. We identified five genes, CCT6, SSB1, ICY1, TIP41, and PBP1 as high-copy suppressors of the tim18 lethality on EtBr-containing medium. As shown in Fig 1, when tim18 cells containing an empty vector were pregrown on medium with EtBr (Fig 1A) and then subcultured onto media lacking EtBr (Fig 1B), the tim18 strain failed to grow. In other words, the EtBr-induced mtDNA loss was incompatible with the lack of the Tim18 protein. However, if the tim18 mutant carried 2µ-URA3 plasmids with CCT6, SSB1, ICY1, TIP41, or PBP1, the cells were able to grow after EtBr treatment nearly as well as tim18 cells with a TIM18-containing plasmid. Although it was previously reported that increased levels of TIM22 partially suppressed the tim18 mutant (KERSCHER et al. 2000 ), we found that 2µ plasmids with TIM22 did not allow tim18 cells to grow without mtDNA (Fig 1). Therefore, our results indicate that the mtDNA dependence of tim18 strains is not due strictly to lower steady-state levels of Tim22p.

View larger version (82K): In this window In a new window Download PPT slide   Figure 1. Cytosolic proteins rescue the mtDNA dependence of tim18 cells. tim18 strain RJ992 was transformed with 2µ-URA3 plasmids carrying TIM18 (pM346), CCT6 (pM350), SSB1 (pM351), ICY1 (pM352), PBP1 (pM353), TIM22 (pJH202; KERSCHER et al. 1997 ), TIP41 (pM345), or empty vector pRS426 (SIKORSKI and HIETER 1989 ). (A) Ura+ transformants were streaked out onto SD-Ura plates containing 25 µg/ml EtBr to induce loss of mtDNA and allowed to grow at 34° for 3 days. (B) To identify strains killed by the EtBr treatment, cells from plate A were streaked to SD-Ura medium lacking EtBr, and the plates were incubated at 34° for 4 days.

Although Tim18p is a mitochondrial IM protein, we noted that none of the proteins encoded by our suppressors have been localized to mitochondria and all appear to be cytosolic proteins. Cct6p is a member of the TriC chaperonin complex and is thought to function in protein folding in the cytosol (LI et al. 1994 ; HARTL and HAYER-HARTL 2002 ). Ssb1p is a member of the Hsp70 chaperone family and has been shown to interact with nascent chains during their synthesis (HUNDLEY et al. 2002 ). While little is known about Icy1p, this protein suppresses a mutation in the Tcp1 protein, which is a partner of Cct6p within the TriC complex (D. URSIC, unpublished observations). TIP41 encodes a cytosolic protein proposed to function as a negative regulator of the yeast TOR-signaling pathway (JACINTO et al. 2001 ). Pbp1p interacts with a (poly)A-binding protein and, like Ssb1p, can be found associated with cytosolic ribosomes (MANGUS et al. 1998 ). Deletions of SSB1, ICY1, TIP41, and PBP1 are viable, while CCT6 is an essential gene (CRAIG and JACOBSEN 1985 ; LI et al. 1994 ; MANGUS et al. 1998 ; JACINTO et al. 2001 ; GIAEVER et al. 2002 ).

In addition to suppressing the mtDNA dependence of tim18 cells, 2µ plasmids carrying SSB1, PBP1, and ICY1 partially rescued the cold sensitivity of the tim18 mutant (C. DUNN, unpublished observations). CCT6- and TIP41-containing plasmids either did not suppress the cold-sensitive phenotype of tim18 or did so very weakly. Why genes such as CCT6 or TIP41 could suppress the mtDNA dependence of tim18 mutants, but not the cold sensitivity, is not clear.

Strains defective in other steps of the mitochondrial protein import pathway are also inviable without mtDNA:
We found that the requirement for mtDNA is not restricted to tim18 cells and that other members of the import machinery are necessary for the viability of petite cells. For example, we found that cells lacking the mitochondrial outer membrane Tom70 receptor protein cannot live without mtDNA. Like tim18 cells, the tom70 mutant failed to grow after EtBr treatment (Fig 2A). We also found that the presence of Tim54p and a fully functional Tim10p, two other components of the TIM22 pathway, are required for petite cell viability (C. DUNN, unpublished observations). Since Tom70p, Tim10p, Tim54p, and Tim18p are all part of the TIM22 pathway, it is possible that dependence on mtDNA is limited to this import route. Supporting this idea, we find that cells with a deficient TIM23 pathway are viable after mtDNA loss, as a strain deleted of the outer membrane Tom20p import receptor and strains containing the mutant Tim23-1 or Ssc1-2 proteins grow after EtBr treatment (C. DUNN and J. EMTAGE, unpublished observations). Nonetheless, further analyses are needed to show that mtDNA dependence is truly specific to one import pathway.

View larger version (46K): In this window In a new window Download PPT slide   Figure 2. tom70 cells require mtDNA when grown on rich medium. Wild-type (WT; RJ868), tim18 (RJ1263), and tom70 (RJ1583) were streaked to SD complete medium + EtBr and grown at 30° for 3 days (not shown). Strains were then streaked to YEPD (A) or SD complete (B) medium lacking EtBr and grown for 3 days at 30°. (C) tom70 strain RJ1583 was transformed with 2µ-URA3 plasmids with CCT6 (pM350), SSB1 (pM351), PBP1 (pM353), TIP41 (pM345), ICY1 (pM352), TIM18 (pM346), TOM70-containing ARS/CEN plasmid pM354, or empty vector pRS426. Ura+ transformants were pregrown on EtBr-containing medium and then streaked onto YEPD plates lacking EtBr.

In contrast to the tim18 mutant, the mtDNA dependence of our tom70 strain was seen only on rich medium. tom70 cells were viable after EtBr treatment when grown on minimal media (Fig 2B). tim18 cells could not grow if forced to lose their mtDNA on either rich or minimal medium (compare A and B in Fig 2). A further indication that the mtDNA requirement for tom70 cells was not as strict as for the tim18 mutant was that a tom70 ⁰ strain derived from the YPH499 background (SIKORSKI and HIETER 1989 ) could lose its mtDNA on both rich and minimal media (C. DUNN, unpublished observations), while the requirement of tim18 cells for mtDNA is not background specific.

For tom70 and other mutants (see below), a few survivor colonies appeared after EtBr treatment, even though the vast majority of cells died. tom70 survivors after EtBr treatment were found to have lost functional mtDNA, since they were no longer viable on nonfermentable medium. Preliminary results suggest that, in two cases, nuclear-encoded mutations allowed tom70 cells to live after mtDNA loss. However, since we tested only two survivors, it is also possible that some colonies simply adapted to mtDNA loss.

We found that our plasmid suppressors of the tim18 mutant also rescued the growth defect of tom70 cells after EtBr treatment. A tom70 strain carrying 2µ-URA3 constructs with CCT6, PBP1, ICY1, or TIP41 were able to grow after mtDNA loss (Fig 2C). For reasons that are not clear, SSB1 only weakly suppressed the tom70 defect. We also noted that while tim18 and tom70 are both petite negative, and both mutants can be rescued by the same set of suppressor-containing plasmids, multiple copies of TIM18 did not bypass the mtDNA requirement of tom70 (Fig 2C). Our observations support the view that a defect in any step in the polytopic protein import pathway, and not the loss of one protein per se, leads to mtDNA dependence.

Cells disrupted in SSB1, PBP1, ICY1, and TIP41 are petite negative:
Since CCT6, SSB1, ICY1, TIP41, and PBP1 were found to suppress the lethality of both tim18 and tom70 after EtBr treatment, we asked if these genes are themselves required for cell viability in the absence of mtDNA. Our results show that Ssb1p, Icy1p, Tip41p, and Pbp1p are required for growth without mtDNA (Fig 3A). Since CCT6 is an essential gene, we were unable to examine the relationship of Cct6p and mtDNA using this approach. Like the tom70 mutant, ssb1, icy1, tip41, and pbp1 cells were viable after EtBr treatment when grown on minimal medium (Fig 3B). Hence the need for these proteins is not as strict as for the Tim18 protein.

View larger version (55K): In this window In a new window Download PPT slide   Figure 3. pbp1, icy1, tip41, and ssb1 mutants are petite negative. Wild-type (WT; RJ868), tim18 (RJ1263), pbp1 (RJ1587), icy1 (RJ1585), tip41 (RJ1584), and ssb1 (RJ1586) were pregrown on EtBr-containing minimal medium and then streaked onto either YEPD (A) or SD complete (B) medium lacking EtBr and grown for 3 days at 30°. (C) Rescue of the mtDNA dependence of ssb1 by tim18 suppressor plasmids. ssb1 strain RJ1586 was transformed with SSB1 (pM351), CCT6 (pM350), PBP1 (pM353), TIP41 (pM345), ICY1 (pM352), TIM18 (pM346), or empty vector pRS426. Ura+ transformants were pregrown on YEPD medium with EtBr and then streaked to YEPD plates lacking EtBr.

The mtDNA dependence of a strain lacking one of the cytosolic suppressors can itself be rescued by multiple copies of the other suppressors. For example, the ssb1 mutant carrying an empty vector cannot grow after EtBr treatment on rich medium (Fig 3C). In contrast, 2µ plasmids with CCT6, PBP1, TIP41, and SSB1 allowed ssb1 cells to grow without mtDNA. We noted that ICY1 did not rescue ssb1. Although the reasons are not apparent, it is possible that Icy1p requires Ssb1p for activity. Similar to our results with tom70, we found that multiple copies of TIM18 did not allow ssb1 cells to grow after EtBr treatment. We speculate that Tim18p, Tom70p, Ssb1p, Icy1p, Tip41p, Pbp1p, and Cct6p all participate in a common pathway and that each protein becomes even more crucial when cells lack mtDNA.

We also found that 2µ plasmids with CCT6, PBP1, TIP41, ICY1, and SSB1 allowed cells lacking mtDNA, but otherwise wild type, to grow at faster rates. Strain RJ1582, which lacks mtDNA, was transformed with each plasmid, and the growth rate of cells was determined (Table 2). All isolated suppressors allowed RJ1582 to grow at a faster rate in comparison to cells carrying an empty vector. These observations provide additional evidence that these proteins play an important role in cells lacking mtDNA.

View this table: In this window In a new window   Table 2. tim18 suppressors increase the growth rate of 0 cells

Slowing protein synthesis lessens the growth defect of tim18 cells:
Our results showed that tom70, ssb1, icy1, tip41, and pbp1 mutants require mtDNA on rich medium, but not on minimal medium. Since the rate of translation is greater when cells are grown on rich media than on minimal media (WEHR and PARKS 1969 ), we wondered if the problems caused by a deficient TIM22 pathway would be alleviated if the rate of protein synthesis were slowed by using sublethal amounts of the translation inhibitor cycloheximide. We were unable to test whether tim18 ⁰ cells can grow in the presence of cycloheximide, because ⁰ cells are quite resistant to the effects of cycloheximide as a result of activation of the pleiotropic drug resistance pathway (HALLSTROM and MOYE-ROWLEY 2000 ). However, we found that cycloheximide suppressed the growth defect of a ⁺ tim18 mutant. As shown in Fig 4, instead of the smooth, round colony morphology seen with wild-type cells, the tim18 mutant forms colonies that are irregular and scallop shaped (Fig 4). Strikingly, if the tim18 mutant is grown on medium containing 0.2 µg/ml cycloheximide, single cells grow into perfectly smooth colonies (Fig 4). In similar studies, slowing protein synthesis with cycloheximide was found to suppress phenotypes associated with defects in the translocation of proteins into the endoplasmic reticulum (OGG and WALTER 1995 ; WILKINSON et al. 2001 ).

View larger version (156K): In this window In a new window Download PPT slide   Figure 4. Inhibiting protein synthesis suppresses the growth defect of tim18 cells lacking mtDNA. Wild type (WT; RJ868) and tim18 (RJ1263) cells were grown on YEPD medium lacking (-CHX) or containing (+CHX) 0.2 mg/ml cycloheximide at 30°.

tim18 cells require a functional respiratory chain:
To pinpoint the reason that lack of mtDNA is incompatible with defects in mitochondrial protein import, we tested whether defects in nuclear-encoded electron transport proteins were tolerated by tim18 cells. We found that the tim18 mutation was lethal in combination with the lack of either cytochrome c₁ or a protein involved in ubiquinone biosynthesis. tim18 cells were crossed to a cyt1 strain or to a coq4 strain. We found that tim18 cyt1 and tim18 coq4 double mutants were initially viable when spores were allowed to germinate on glucose-containing medium. However, the double mutants failed to grow when they were subsequently streaked out for single colonies on YEPD medium (Fig 5). Why the lethality of tim18 cyt1 and tim18 coq4 mutants was delayed is not clear. cyt1 and coq4 deletion strains do not quickly lose mtDNA, as tested by DAPI staining of nucleoids in coq4 and cyt1 strains and by mating individual coq4 and cyt1 colonies to ⁰ tester strains. Therefore, our observations indicate that the mtDNA dependence of tim18 cells is due to a general defect in electron transport.

View larger version (89K): In this window In a new window Download PPT slide   Figure 5. tim18 is lethal in combination with nuclear-encoded defects in respiration. After tetrad dissection on YEPD at 34°, meiotic segregants were streaked onto YEPD medium and allowed to grow for 3 days at 30°. (A) Cells from the four spores of a tetratype tetrad produced by the tim18 (RJ1263) x coq4 (RJ1606) cross. The tetrad used for the analysis is indicated by the gray rectangle. (B) Cells from the four spores of a tetratype tetrad produced by the tim18 (RJ1263) x cyt1 (RJ1605). The tetrad used for the analysis is indicated by the shaded rectangle.

Multiple copies of CCT6, SSB1, ICY1, TIP41, and PBP1 rescue the mtDNA dependence of atp2 mutants, but not the need for ATP in the matrix:
The results above raise the possibility that the lethality associated with loss of mtDNA may be due to a decrease in the electrochemical potential across the IM. When electron transport is compromised by loss of mtDNA, the IM potential is thought to be produced by the transport of ATP across the IM by the ATP/ADP carrier proteins, followed by ATP hydrolysis by the F₁ ATPase (CHEN and CLARK-WALKER 2000 ). Consistent with this view, atp2 mutants, lacking the ß-subunit of the F₁ ATPase, and op1 mutants, lacking a fully functional copy of the major ATP/ADP carrier Aac2p, are unable to grow without mtDNA (KOVACOVA et al. 1968 ; CHEN and CLARK-WALKER 1999 ).

We find that the high-copy suppressors of tim18 also rescued the petite-negative phenotype of atp2 mutants. While the atp2 mutant with an empty vector or with 2µ-TIM18 does not grow without mtDNA, atp2 cells carrying CCT6, SSB1, PBP1, TIP41, or ICY1-containing plasmids grow at or near wild-type rates after treatment with EtBr (Fig 6A). Thus, the tim18 suppressors also rescue the petite-negative phenotype of cells lacking a protein known to be important for generating the IM potential, the ATP2-encoded ATPase subunit. These results therefore support our view that increased amounts of Ssb1p, Icy1p, Tip41p, Pbp1p, and Cct6p all alleviate the problems caused by a decreased mitochondrial potential in petite cells. We note that the mtDNA dependence of atp2 mutants is greatly reduced when cells are grown on minimal medium (Fig 6C), suggesting that other factors in addition to the F₁ ATPase can help generate the IM potential in cells lacking mtDNA.

View larger version (56K): In this window In a new window Download PPT slide   Figure 6. (A) The petite-negative phenotype of the atp2 mutant is rescued by tim18 suppressors. atp2 strain RJ1577 was transformed with 2µ-URA3 plasmids containing CCT6 (pM350), SSB1 (pM351), PBP1 (pM353), TIP41 (pM345), ICY1 (pM352), TIM18 (pM346), and pRS426. 0 strain RJ1582 transformed with pRS426 is also shown. Cells were pregrown on YEPD medium with EtBr and then streaked to YEPD plates lacking EtBr and grown for 4 days at 30°. (B) op1 cells, lacking functional Aac2 protein, remain mtDNA dependent. op1 strain RJ1581 was transformed with 2µ-URA3 plasmids containing CCT6 (pM350), SSB1 (pM351), PBP1 (pM353), TIP41 (pM345), ICY1 (pM352), and TIM18 (pM346), pregrown on YEPD medium with EtBr and then streaked to YEPD medium lacking EtBr for 5 days at 30°. (C) atp2 cells lacking mtDNA grow slowly on minimal medium, but fail to grow on rich medium. Wild type strain RJ868 and atp2 strain RJ1577 were pregrown on either SD complete or YEPD medium containing EtBr for 3 days at 30° and then subcultured on the same medium lacking EtBr for 4 days at 30°.

The inner membrane protease Yme1p has been shown to be important for the growth of petite cells (THORSNESS et al. 1993 ). YME1 genetically interacts with the F₁ ATPase and has been shown to increase the electrochemical potential in ⁰ mitochondria (WEBER et al. 1995 ; KOMINSKY and THORSNESS 2000 ; KOMINSKY et al. 2002 ). We asked whether YME1 might also interact genetically with TIM18 or with any of its isolated suppressors. However, a strain lacking both Tim18p and Yme1p was viable (C. DUNN, unpublished results) and none of our isolated high-copy suppressors rescued the severe growth defect of a yme1 ⁰ strain. Therefore, while both genes are required for growth of petite cells, no direct genetic link has yet been demonstrated between YME1 and TIM18.

In addition, in contrast to atp2, the mtDNA dependence of op1 mutants is not rescued by our suppressor-containing plasmids. An op1 strain carrying CCT6-, SSB1-, PBP1-, TIP41-, or ICY1-containing plasmids failed to grow at wild-type rates after treatment with EtBr (Fig 6B). Cells lacking mtDNA are acutely reliant upon the Aac2 protein, indicating an absolute need for mitochondrial ATP/ADP transport in petite cells.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To learn more about the role of the Tim18 protein, a member of the TIM22 inner membrane import complex, we isolated high-copy suppressors of the inability of tim18 mutants to grow in the absence of mtDNA. However, instead of finding new inner membrane components of the TIM22 complex, all of our suppressor-containing plasmids encode proteins thought to be located within the cytosol, such as the Cct6, Ssb1, Icy1, Tip41, and Pbp1 proteins.

We found that dependence upon mtDNA is not limited to the tim18 mutant; mutations in genes encoding other mitochondrial protein import components can also generate a petite-negative phenotype. For example, cells lacking the TIM22 complex member Tim54p were unable to grow on medium that selects for mtDNA loss. In addition, a strain containing the tim10-2 allele, encoding a defective intermembrane space member of the TIM22 complex, is petite negative. Furthermore, a tom70 disruption mutant lacking the outer membrane Tom70p receptor was inviable after treatment with ethidium bromide. Since Tim18p, Tim54p, Tim10p, and Tom70p all function in the TIM22 pathway, which is required for the import of polytopic IM proteins, it is possible that this pathway is particularly sensitive to loss of mtDNA. Consistent with this possibility, we find that mutations in three members of the TIM23 pathway, which is required primarily for the import of presequence-containing proteins into the matrix, are not lethal in combination with mtDNA loss. A tom20 strain, lacking the Tom20p receptor, and the tim23-1 mutant, defective in the inner membrane TIM23 complex protein Tim23p, are completely viable following growth on ethidium-bromide-containing medium. Moreover, a ssc1-2 mutant, deficient in translocation through the TIM23 complex, can live in the absence of mtDNA. However, further studies are needed to determine if mtDNA dependence is truly specific to deficiencies within the TIM22 pathway.

Why are defects in mitochondrial protein import incompatible with mtDNA loss? Mitochondrial DNA encodes for proteins that function in electron transport. We argue that the lack of mitochondrial ATP synthesis is not the cause of the mtDNA dependence of import mutants, since tim18, tim54, tim10-2, and tom70 cells lacking mtDNA are dead even on glucose-containing medium. On glucose, sufficient levels of ATP are produced by glycolysis and mtDNA is not required (CHEN and CLARK-WALKER 2000 ). Instead, we suggest that the petite-negative phenotype of the import mutants is caused by a lowered IM potential resulting from defective electron transport in cells lacking mtDNA. Supporting this idea, we found that tim18 is lethal in combination with mutations in two different nuclear-encoded components required for electron transport, cytochrome c₁ and Coq4p (SCHNEIDER and GUARENTE 1991 ; BELOGRUDOV et al. 2001 ). In addition, suppressors of tim18 also suppress the petite-negative phenotype of a strain lacking ATP2, encoding a protein believed to play a role in generating mitochondrial potential in the absence of mtDNA (GIRAUD and VELOURS 1997 ; CHEN and CLARK-WALKER 1999 ). Since the mitochondrial potential is essential for activity of the TIM22 complex, we suggest that when IM potential is lowered, changes in the efficiency of import caused by the loss of nonessential proteins, such as Tim18p, Tim54p, or Tom70p, or conditional defects in the function of an essential protein, such as Tim10p, become magnified. We propose that under these conditions imported proteins build up outside the mitochondria and are toxic to the yeast cell. Our model is supported by the properties of the high-copy suppressors of the petite-negative phenotype of the tim18 mutant.

Our suppressors seem to fall into at least two categories: cytosolic chaperones and proteins that may affect protein synthesis. SSB1 and CCT6 encode proteins known to function as cytosolic chaperones. Ssb1p is a member of the Hsp70 family (BOORSTEIN et al. 1994 ) and Cct6p is part of the heteroligomeric TriC chaperonin complex (LI et al. 1994 ). One explanation for how high-copy plasmids encoding SSB1 and CCT6 suppress the mtDNA dependence of some import mutants is that cytosolic chaperones can become limiting when mitochondrial protein import is compromised. Since mitochondrial precursor proteins bind to chaperones prior to their import into mitochondria (BEDDOE and LITHGOW 2002 ), precursors that build up outside the mitochondria may compete with other, essential cellular processes for chaperone activity and therefore lead to cell death. Under these conditions, increasing the amount of a limiting chaperone would restore viability.

Cytosolic Hsp70 proteins have been shown to facilitate the import of several different mitochondrial precursor proteins (BEDDOE and LITHGOW 2002 ), and it has been recently reported that Hsp70 proteins directly transfer some preproteins to the Tom70p receptor (YOUNG et al. 2003 ). Thus, it is reasonable to assume that chaperones, such as Ssb1p, might bind mitochondrial precursors accumulating due to a defect in import. Supporting this idea, the bacterial Hsp70 protein dnaK has been found to tightly bind to membrane protein precursors when the activity of the Sec translocon is diminished (QI et al. 2002 ). However, since Ssb1p is only one of several cytosolic Hsp70 proteins, it is not clear why other Hsp70 family members were not isolated in our genetic screen. Furthermore, we found that 2µ plasmids carrying SSB2 did not suppress the mtDNA dependence of tim18 mutants (C. DUNN, unpublished observations). Since Ssb1p differs at only four amino acid residues from Ssb2p, further studies are needed to determine if Ssb1p truly plays some unique role in mitochondrial protein import.

In contrast to Hsp70 proteins, there is no compelling evidence that the TriC chaperonin complex plays a direct role in mitochondrial protein import. However, TriC has been shown to interact with peroxisomal (PAUSE et al. 1997 ) and endoplasmic reticulum (ER; PLATH and RAPOPORT 2000 ) proteins prior to their import, so it would not be surprising if the chaperonin complex did facilitate the import of mitochondrial proteins. On the other hand, it is possible that the abnormal accumulation of mitochondrial proteins recruits chaperones not normally associated with protein import. Most of the substrates for the TIM22 pathway are polytopic membrane proteins, and these hydrophobic proteins would be particularly prone to aggregation in the cytosol. However, since TriC is composed of eight different subunits (LIN and SHERMAN 1997 ; LIOU and WILLISON 1997 ), it is curious that only one TriC complex member was identified in our suppressor screen. Perhaps overexpressed Cct6p provides some chaperone activity in the absence of the other TriC subunits. Alternatively, Cct6p may have a more indirect role in suppressing problems with protein import, possibly by acting in a signaling pathway (SCHMIDT et al. 1996 ).

The ICY1 gene was also identified as a high-copy suppressor in our studies. Since ICY1 genetically interacts with the TriC complex, suppressing a mutation in the TriC subunit Cct1p (D. URSIC, unpublished observations), we include Icy1p in the same category with the chaperones Ssb1p and Cct6p. Although the function of Icy1p is not known, it is interesting to note that the expression of ICY1 increases when mtDNA is lost (EPSTEIN et al. 2001 ).

Proteins and conditions that appear to decrease the rate of protein synthesis make up our second category of suppressors of the petite-negative tim18 mutant. For example, high-copy TIP41 was isolated in our screen. Tip41p is a negative regulator of the TOR-signaling pathway (JACINTO et al. 2001 ), and the TOR pathway functions in sensing nutrient levels and adjusting translation accordingly (SCHMELZLE and HALL 2000 ). Thus, it is possible that overexpression of Tip41p decreases translation. We find that cycloheximide suppresses a growth defect of the tim18 strain, also indicating that slowing protein synthesis can ameliorate a defect in protein import. Moreover, decreasing translation with cycloheximide or by mutation has been shown to rescue phenotypes associated with defective protein translocation into the ER (OGG and WALTER 1995 ; WILKINSON et al. 2001 ) and bacterial secretion (LEE and BECKWITH 1986 ). The expression of many components of the cytosolic translational machinery are downregulated upon loss of mtDNA (EPSTEIN et al. 2001 ), suggesting that modulating protein synthesis is a general cellular adaptation to mtDNA loss. If our model is correct, decreased translation would prevent the toxic accumulation of unimported mitochondrial proteins.

Although we have suggested that Ssb1p, Cct6p, and Icy1p suppress defects in protein import by binding to accumulated precursor proteins, we are also open to the possibility that overexpression of these proteins may instead regulate translation. For example, both Ssb1p (HUNDLEY et al. 2002 ) and the TriC complex (MCCALLUM et al. 2000 ) are thought to bind to nascent chains. Perhaps inappropriate amounts of Ssb1p, Cct6p, and/or Icy1p interact directly with the ribosome and slow protein synthesis.

PBP1 was also isolated as a suppressor in our genetic screen. Pbp1p interacts with a poly(A)-binding protein (MANGUS et al. 1998 ) and is found, like Ssb1p, in the nucleus on ribosomes and in the cytosol (MANGUS et al. 1998 ). As suggested for Ssb1p, increased levels of Pbp1p could rescue an import defect by either affecting protein synthesis or functioning as a chaperone. Strikingly, PBP1 was first isolated in a screen that also identified Tim10p and Tim12p (WALDHERR et al. 1993 ), proteins known to be part of the TIM22 import pathway. The actual role of Pbp1p, however, awaits further analysis.

Although we favor the idea that multiple copies of CCT6, SSB1, ICY1, TIP41, and PBP1 prevent toxic accumulation of mitochondrial precursor proteins, there are additional possibilities that could explain their suppressor effects. For example, one or more of the suppressors could in some way act to increase the mitochondrial potential when overexpressed, thus allowing mitochondrial import to occur more efficiently in cells lacking mtDNA. Evidence for this possibility is lacking since staining of ⁰ cells containing the high-copy suppressors with the potentially dependent dye DiOC₆ did not show increased mitochondrial fluorescence (C. DUNN, unpublished observations). Alternatively, the suppressors may modulate a mitochondria-to-nucleus signaling pathway, such as the retrograde response pathway (LIU et al. 2001 ; CRESPO et al. 2002 ). It is also possible that the suppressors all act to increase the import or stability of a single mitochondrial component. Future experiments are clearly needed to determine the mechanism by which CCT6, SSB1, ICY1, TIP41, and PBP1 act in rescuing petite-negative cells.

It is interesting to note that not only do SSB1, ICY1, TIP41, and PBP1 suppress the mtDNA dependence of some import mutants, but also disruption of any of these genes produces cells that are themselves petite negative. Our studies have therefore identified a genetic pathway, starting with the mitochondria IM proteins Tim18p and Tim54p, proceeding through the intermembrane space (Tim10p) and the outer membrane (Tom70p), and ending in the cytosol with the Cct6, Ssb1, Icy1, Tip41, and Pbp1 proteins. All of these components are required for yeast cell growth in the absence of mtDNA.

Our initial goal in the isolation of suppressors of tim18 was to further understand the role that Tim18p plays in import. Because tim18 mutants are dead in cells lacking a functional respiratory chain, we originally thought that Tim18p functions at a potentially dependent step in import. At first glance, our observations that mutants defective in nonmitochondrial proteins are also dependent upon mtDNA seem to argue against this possibility. However, we found that tim18 mutants are more sensitive to mtDNA loss than are most of our other mutants. tim18 cells are unable to grow on either rich or minimal medium containing ethidium bromide, whereas cells carrying tom70, ssb1, icy1, tip41, and pbp1 mutations are dead only on rich medium following mtDNA loss. Since strains grown on rich medium exhibit increased protein synthesis rates in comparison to those grown on minimal medium (WEHR and PARKS 1969 ), these observations further support our conclusion that slowing translation helps cells cope with the loss of mtDNA. Nonetheless, the exquisite mtDNA dependence of tim18 mutants again raises the possibility that Tim18p may truly act in a potentially dependent manner. Studies to further examine this possibility are underway.

	ACKNOWLEDGMENTS

We thank Gulayse Ince, Hiromi Sesaki, Alyson Aiken Hobbs, Kara Cerveny, Matt Youngman, Heidi Hoard-Fruchey, Mariana Melani, Sheryl Southard, Oliver Kerscher, and Carolyn Machamer for comments on the manuscript. We thank Doris Ursic for communicating unpublished results; Doris Ursic, Peter Thorsness, Betty Craig, and Klaus Pfanner for yeast strains; and Phil Hieter for the 2µ-URA3 library. This work was supported by U.S. Public Health Service grant RO1-GM46803 to R.E.J. and a National Institutes of Health Predoctoral Training Grant 5T32GM07445 to C.D.D.

Manuscript received January 27, 2003; Accepted for publication April 23, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ADAMS, A., D. GOTTSCHLING, C. KAISER and T. STEARNS, 1997 Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Plainview, NY.

BEDDOE, T. and T. LITHGOW, 2002  Delivery of nascent polypeptides to the mitochondrial surface. Biochim. Biophys. Acta 1592:35-39.[Medline]

BELOGRUDOV, G. I., P. T. LEE, T. JONASSEN, A. Y. HSU, and P. GIN et al., 2001  Yeast COQ4 encodes a mitochondrial protein required for coenzyme Q synthesis. Arch. Biochem. Biophys. 392:48-58.[Medline]

BOORSTEIN, W. R., T. ZIEGELHOFFER, and E. A. CRAIG, 1994  Molecular evolution of the HSP70 multigene family. J. Mol. Evol. 38:1-17.[Medline]

BRACHMANN, C. B., A. DAVIES, G. J. COST, E. CAPUTO, and J. LI et al., 1998  Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115-132.[Medline]

CHEN, X. J. and G. D. CLARK-WALKER, 1999  Alpha and beta subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae. Mol. Gen. Genet. 262:898-908.[Medline]

CHEN, X. J. and G. D. CLARK-WALKER, 2000  The petite mutation in yeasts: 50 years on. Int. Rev. Cytol. 194:197-238.[Medline]

CRAIG, E. A. and K. JACOBSEN, 1985  Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures. Mol. Cell. Biol. 5:3517-3524.[Abstract/Free Full Text]

CRESPO, J. L., T. POWERS, B. FOWLER, and M. N. HALL, 2002  The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. Proc. Natl. Acad. Sci. USA 99:6784-6789.[Abstract/Free Full Text]

DUPONT, C. H., J. P. MAZAT, and B. GUERIN, 1985  The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from rho + and rho degree strains of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 132:1116-1123.[Medline]

EILERS, M., W. OPPLIGER, and G. SCHATZ, 1987  Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J. 6:1073-1077.[Medline]

EPSTEIN, C. B., J. A. WADDLE, W. HALE, IV, V. DAVE, and J. THORNTON et al., 2001  Genome-wide responses to mitochondrial dysfunction. Mol. Biol. Cell 12:297-308.[Abstract/Free Full Text]

GAKH, O., P. CAVADINI, and G. ISAYA, 2002  Mitochondrial processing peptidases. Biochim. Biophys. Acta 1592:63-77.[Medline]

GEISSLER, A., A. CHACINSKA, K. N. TRUSCOTT, N. WIEDEMANN, and K. BRANDNER et al., 2002  The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel. Cell 111:507-518.[Medline]

GIAEVER, G., A. M. CHU, L. NI, C. CONNELLY, and L. RILES et al., 2002  Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387-391.[Medline]

GIRAUD, M. F. and J. VELOURS, 1997  The absence of the mitochondrial ATP synthase delta subunit promotes a slow growth phenotype of rho- yeast cells by a lack of assembly of the catalytic sector F1. Eur. J. Biochem. 245:813-818.[Medline]

GOLDRING, E. S., L. I. GROSSMAN, D. KRUPNICK, D. R. CRYER, and J. MARMUR, 1970  The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J. Mol. Biol. 52:323-335.[Medline]

HALLSTROM, T. C. and W. S. MOYE-ROWLEY, 2000  Multiple signals from dysfunctional mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces cerevisiae. J. Biol. Chem. 275:37347-37356.[Abstract/Free Full Text]

HARTL, F. U. and M. HAYER-HARTL, 2002  Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295:1852-1858.[Abstract/Free Full Text]

HOFFMAN, C. S. and F. WINSTON, 1987  A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267-272.[Medline]

HOOGENRAAD, N. J., L. A. WARD, and M. T. RYAN, 2002  Import and assembly of proteins into mitochondria of mammalian cells. Biochim. Biophys. Acta 1592:97-105.[Medline]

HUNDLEY, H., H. EISENMAN, W. WALTER, T. EVANS, and Y. HOTOKEZAKA et al., 2002  The in vivo function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain. Proc. Natl. Acad. Sci. USA 99:4203-4208.[Abstract/Free Full Text]

JACINTO, E., B. GUO, K. T. ARNDT, T. SCHMELZLE, and M. N. HALL, 2001  TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol. Cell 8:1017-1026.[Medline]

JENSEN, R. E. and C. D. DUNN, 2002  Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocons. Biochim. Biophys. Acta 1592:25-34.[Medline]

KERSCHER, O., J. HOLDER, M. SRINIVASAN, R. S. LEUNG, and R. E. JENSEN, 1997  The Tim54p-Tim22p complex mediates insertion of proteins into the mitochondrial inner membrane. J. Cell Biol. 139:1663-1675.[Abstract/Free Full Text]

KERSCHER, O., N. B. SEPURI, and R. E. JENSEN, 2000  Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane. Mol. Biol. Cell 11:103-116.[Abstract/Free Full Text]

KOEHLER, C. M., S. MERCHANT, and G. SCHATZ, 1999  How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem. Sci. 24:428-432.[Medline]

KOEHLER, C. M., M. P. MURPHY, N. A. BALLY, D. LEUENBERGER, and W. OPPLIGER et al., 2000  Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane. Mol. Cell. Biol. 20:1187-1193.[Abstract/Free Full Text]

KOMINSKY, D. J. and P. E. THORSNESS, 2000  Expression of the Saccharomyces cerevisiae gene YME1 in the petite-negative yeast Schizosaccharomyces pombe converts it to petite-positive. Genetics 154:147-154.[Abstract/Free Full Text]

KOMINSKY, D. J., M. P. BROWNSON, D. L. UPDIKE, and P. E. THORSNESS, 2002  Genetic and biochemical basis for viability of yeast lacking mitochondrial genomes. Genetics 162:1595-1604.[Abstract/Free Full Text]

KOVACOVA, V., J. IRMLEROVA, and L. KOVAC, 1968  Oxidative phosphorylation in yeast. IV. Combination of a nuclear mutation affecting oxidative phosphorylation with cytoplasmic mutation to respiratory deficiency. Biochim. Biophys. Acta 162:157-163.[Medline]

KOVERMANN, P., K. N. TRUSCOTT, B. GUIARD, P. REHLING, and N. B. SEPURI et al., 2002  Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel. Mol. Cell 9:363-373.[Medline]

LEE, C. A. and J. BECKWITH, 1986  Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. J. Bacteriol. 166:878-883.[Abstract/Free Full Text]

LI, W. Z., P. LIN, J. FRYDMAN, T. R. BOAL, and T. S. CARDILLO et al., 1994  Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast. J. Biol. Chem. 269:18616-18622.[Abstract/Free Full Text]

LIN, P. and F. SHERMAN, 1997  The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex. Proc. Natl. Acad. Sci. USA 94:10780-10785.[Abstract/Free Full Text]

LIOU, A. K. and K. R. WILLISON, 1997  Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes. EMBO J. 16:4311-4316.[Medline]

LIU, Z., T. SEKITO, C. B. EPSTEIN, and R. A. BUTOW, 2001  RTG-dependent mitochondria to nucleus signaling is negatively regulated by the seven WD-repeat protein Lst8p. EMBO J. 20:7209-7219.[Medline]

MANGUS, D. A., N. AMRANI, and A. JACOBSON, 1998  Pbp1p, a factor interacting with Saccharomyces cerevisiae poly(A)-binding protein, regulates polyadenylation. Mol. Cell. Biol. 18:7383-7396.[Abstract/Free Full Text]

MARTIN, J., K. MAHLKE, and N. PFANNER, 1991  Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences. J. Biol. Chem. 266:18051-18057.[Abstract/Free Full Text]

MCCALLUM, C. D., H. DO, A. E. JOHNSON, and J. FRYDMAN, 2000  The interaction of the chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) with ribosome-bound nascent chains examined using photo-cross-linking. J. Cell Biol. 149:591-602.[Abstract/Free Full Text]

NEUPERT, W. and M. BRUNNER, 2002  The protein import motor of mitochondria. Nat. Rev. Mol. Cell Biol. 3:555-565.[Medline]

OGG, S. C. and P. WALTER, 1995  SRP samples nascent chains for the presence of signal sequences by interacting with ribosomes at a discrete step during translation elongation. Cell 81:1075-1084.[Medline]

PAUSE, B., P. DIESTELKOTTER, H. HEID, and W. W. JUST, 1997  Cytosolic factors mediate protein insertion into the peroxisomal membrane. FEBS Lett. 414:95-98.[Medline]

PFANNER, N. and A. CHACINSKA, 2002  The mitochondrial import machinery: preprotein-conducting channels with binding sites for presequences. Biochim. Biophys. Acta 1592:15-24.[Medline]

PLATH, K. and T. A. RAPOPORT, 2000  Spontaneous release of cytosolic proteins from posttranslational substrates before their transport into the endoplasmic reticulum. J. Cell Biol. 151:167-178.[Abstract/Free Full Text]

QI, H. Y., J. B. HYNDMAN, and H. D. BERNSTEIN, 2002  DnaK promotes the selective export of outer membrane protein precursors in SecA-deficient Escherichia coli. J. Biol. Chem. 277:51077-51083.[Abstract/Free Full Text]

RINE, J., 1991  Gene overexpression in studies of Saccharomyces cerevisae.. Methods Enzymol. 194:239-251.[Medline]

SCHLEYER, M., B. SCHMIDT, and W. NEUPERT, 1982  Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur. J. Biochem. 125:109-116.[Medline]

SCHMELZLE, T. and M. N. HALL, 2000  TOR, a central controller of cell growth. Cell 103:253-262.[Medline]

SCHMIDT, A., J. KUNZ, and M. N. HALL, 1996  TOR2 is required for organization of the actin cytoskeleton in yeast. Proc. Natl. Acad. Sci. USA 93:13780-13785.[Abstract/Free Full Text]

SCHNEIDER, J. C. and L. GUARENTE, 1991  Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4. Mol. Cell. Biol. 11:4934-4942.[Abstract/Free Full Text]

SIKORSKI, R. S. and P. HIETER, 1989  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27.[Abstract/Free Full Text]