In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins.

To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.

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Mitochondrial targeting of yeast apoiso-1-cytochrome c is mediated through functionally independent structural domains

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5763-5771.1990 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5763-5771

Author(s):

S H Nye ◽

R C Scarpulla

Keyword(s):

Cytochrome C ◽

Mitochondrial Membrane ◽

Structural Information ◽

Alpha Helix ◽

Initial Step ◽

Intermembrane Space ◽

Mitochondrial Targeting ◽

Structural Determinants ◽

Amino Terminal

An iso-1-cytochrome c-chloramphenicol acetyltransferase fusion protein (iso-1/CAT) was expressed in Saccharomyces cerevisiae and used to delineate two stages in the cytochrome c import pathway in vivo (S. H. Nye and R. C. Scarpulla, Mol. Cell. Biol. 10:5753-5762, 1990 [this issue]). Fusion proteins with the CAT reporter domain in its native conformation were arrested at the initial stage of mitochondrial membrane recognition and insertion. In contrast, those with a deletional disruption of the CAT moiety were relieved of this block and allowed to translocate to the intermembrane space, where they functioned in respiratory electron transfer. In the present study, iso-1/CAT was used to map structural determinants in apoiso-1-cytochrome c involved in the initial step of targeting to the mitochondrial membrane. Carboxy-terminal deletions revealed that one of these determinants consisted of the amino-terminal 68 residues. Deletion mutations either within or at the ends of this determinant destroyed mitochondrial targeting activity, suggesting that functionally important information spans the length of this fragment. Disruption of an alpha-helix near the amino terminus by a helix-breaking proline substitution for leucine 14 also eliminated the targeting activity of the 1 to 68 determinant, suggesting a contribution from this structure. A second, functionally independent targeting determinant was found in the carboxy half of the apoprotein between residues 68 and 85. This determinant coincided with a stretch of 11 residues that are invariant in nearly 100 eucaryotic cytochromes c. Therefore, in lieu of an amino-terminal presequence, apocytochrome c has redundant structural information located in both the amino and carboxy halves of the molecule that can function independently to specify mitochondrial targeting and membrane insertion in vivo.

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Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5487 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5487-5496 ◽

Cited By ~ 105

Author(s):

M E Dumont ◽

T S Cardillo ◽

M K Hayes ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Mitochondrial Membrane ◽

Intermembrane Space ◽

Inner Mitochondrial Membrane ◽

Yeast Saccharomyces Cerevisiae ◽

Apocytochrome C ◽

Cytochrome C Heme Lyase

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

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Mitochondrial targeting of yeast apoiso-1-cytochrome c is mediated through functionally independent structural domains.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5763 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5763-5771 ◽

Cited By ~ 19

Author(s):

S H Nye ◽

R C Scarpulla

Keyword(s):

Cytochrome C ◽

Mitochondrial Membrane ◽

Structural Information ◽

Alpha Helix ◽

Initial Step ◽

Intermembrane Space ◽

Mitochondrial Targeting ◽

Structural Determinants ◽

Amino Terminal

An iso-1-cytochrome c-chloramphenicol acetyltransferase fusion protein (iso-1/CAT) was expressed in Saccharomyces cerevisiae and used to delineate two stages in the cytochrome c import pathway in vivo (S. H. Nye and R. C. Scarpulla, Mol. Cell. Biol. 10:5753-5762, 1990 [this issue]). Fusion proteins with the CAT reporter domain in its native conformation were arrested at the initial stage of mitochondrial membrane recognition and insertion. In contrast, those with a deletional disruption of the CAT moiety were relieved of this block and allowed to translocate to the intermembrane space, where they functioned in respiratory electron transfer. In the present study, iso-1/CAT was used to map structural determinants in apoiso-1-cytochrome c involved in the initial step of targeting to the mitochondrial membrane. Carboxy-terminal deletions revealed that one of these determinants consisted of the amino-terminal 68 residues. Deletion mutations either within or at the ends of this determinant destroyed mitochondrial targeting activity, suggesting that functionally important information spans the length of this fragment. Disruption of an alpha-helix near the amino terminus by a helix-breaking proline substitution for leucine 14 also eliminated the targeting activity of the 1 to 68 determinant, suggesting a contribution from this structure. A second, functionally independent targeting determinant was found in the carboxy half of the apoprotein between residues 68 and 85. This determinant coincided with a stretch of 11 residues that are invariant in nearly 100 eucaryotic cytochromes c. Therefore, in lieu of an amino-terminal presequence, apocytochrome c has redundant structural information located in both the amino and carboxy halves of the molecule that can function independently to specify mitochondrial targeting and membrane insertion in vivo.

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Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5487-5496.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5487-5496

Author(s):

M E Dumont ◽

T S Cardillo ◽

M K Hayes ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Mitochondrial Membrane ◽

Intermembrane Space ◽

Inner Mitochondrial Membrane ◽

Yeast Saccharomyces Cerevisiae ◽

Apocytochrome C ◽

Cytochrome C Heme Lyase

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

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Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.

The Journal of Cell Biology ◽

10.1083/jcb.123.1.119 ◽

1993 ◽

Vol 123 (1) ◽

pp. 119-126 ◽

Cited By ~ 111

Author(s):

W Voos ◽

B D Gambill ◽

B Guiard ◽

N Pfanner ◽

E A Craig

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Fusion Protein ◽

Fusion Proteins ◽

Dual Role ◽

Intermembrane Space ◽

Heme Binding ◽

Membrane Translocation ◽

Amino Terminal ◽

The Matrix

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

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Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

Biochemical Journal ◽

10.1042/bj3280669 ◽

1997 ◽

Vol 328 (2) ◽

pp. 669-675 ◽

Cited By ~ 17

Author(s):

L. Tamara DOERING ◽

Randy SCHEKMAN

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Proteins ◽

Attachment Site ◽

Gpi Anchor ◽

Mating Pheromone ◽

Peptide Substrates ◽

In Vitro System ◽

Glycosyl Phosphatidylinositol

The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.

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RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach

Molecular and Cellular Biology ◽

10.1128/mcb.22.20.6979-6992.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 6979-6992 ◽

Cited By ~ 357

Author(s):

Nevan J. Krogan ◽

Minkyu Kim ◽

Seong Hoon Ahn ◽

Guoqing Zhong ◽

Michael S. Kobor ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

The Novel ◽

Coding Region ◽

Elongation Factors ◽

Uncharacterized Protein ◽

Proteomics Approach

ABSTRACT To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3′ end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

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Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant

Molecular and Cellular Biology ◽

10.1128/mcb.4.7.1393-1401.1984 ◽

1984 ◽

Vol 4 (7) ◽

pp. 1393-1401

Author(s):

B Errede ◽

T S Cardillo ◽

M A Teague ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Transposable Element ◽

Yeast Cells ◽

Yeast Mutant ◽

Yeast Saccharomyces Cerevisiae ◽

Specific Restriction ◽

Restriction Endonuclease Cleavage

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.

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