The conformational states of cytochrome c inside intact and Ca(2+)-exposed mitochondria have been investigated using resonance Raman spectroscopy. Intact and swelling bovine heart and rat liver mitochondria were examined with an excitation wavelength (413.1 nm) in resonance with the Soret transition of ferrous cytochrome c. The different b- to c-type cytochrome concentration ratio in mitochondria from two different tissues was used to help assign the Raman spectral components. Resonance Raman spectra were also recorded for mitochondria fractions (supernatants and pellets) obtained from swollen (Ca(2+)-exposed) mitochondria after differential centrifugation. The results illustrate that cytochrome c has an altered vibrational spectrum in solution, in intact, and in swollen mitochondria. When cytochrome c is released from mitochondria, its Raman spectrum becomes identical to that of ferrous cytochrome c in solution. The spectra of mitochondrial pellets indicate that a small amount of structurally modified cytochrome c remains associated with the heavy membrane fraction. Indeed, spectroscopic shifts in the low-frequency fingerprint and the high-frequency marker-band regions suggest that membrane binding leads to a partial opening of the heme pocket and an alteration of the heme thioether bonds. The results support the conclusion that most cytochrome c molecules in mitochondria are membrane-bound and that the cytochrome c structure changes upon binding. Furthermore, changes in the resonance Raman active mode located at 675 cm(-)(1) in the spectra of intact, swollen, and fractionated mitochondria indicate that b-type cytochromes may also undergo structural alterations during mitochondrial swelling and disruption.

Yeast mitochondrial phosphate transport activity has been reconstituted from the import receptor (MIR) expressed as inclusion bodies in Escherichia coli. This result undermines the suggestion [Murakami, H., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3358-3362] that the MIR has been misidentified as the phosphate transport protein (PTP). PTP was solubilized with N-lauroylsarcosinate and Triton X-100 and purified with a yield of about 2 mg/L of induced bacterial culture. This PTP, reconstituted in liposomes, catalyzes phosphate uptake with a Vmax [24.5 degrees C, net (zero trans), pHi = 8.0, pHe = 6.8] of 0.61 mmol of phosphate min-1 (mg of PTP)-1 and a Km of 1.30 mM. This Vmax is higher and the Km about the same as that obtained with PTP purified from mitochondria. Replacement of Thr43 and Ile141 by other amino acids results in three types of PTP: (a) 2.5-5.0% Vmax of wild-type PTP (PTPwt) (Thr43Cys; Thr43Ser; Ile141Cys), (b) < 0.1% Vmax (detection limit of assay) of PTPwt (Thr43Ala; Thr43Asp), and (c) proton transport uncoupled from phosphate transport (Ile141Cys). Km changes are not significant. Activity of Thr43Cys confirms results obtained with mitochondrially expressed protein. Thus, yeast PTP requires Thr43 and mammalian PTP the similarly located Cys42 for high transport activity. Thr43 and Ile141 are each situated between two basic residues (LysThrArg vs ArgIleArg). Cys substitutions in either of these positions confer the same high N-ethylmaleimide sensitivity to the yeast PTPwt as displayed by the mammalian PTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Wild type and mutant phosphate carriers (PIC) fromSaccharomyces cerevisiae mitochondria were expressed in Escherichia coli as inclusion bodies, solubilized, purified, and optimally reconstituted into liposomal membranes. This PIC can function as coupled antiport (P i ؊ / P i ؊ antiport and P i ؊ net transport, i.e. P i ؊ /OH ؊ antiport) and uncoupled uniport (mercuric chloride-induced P i ؊ efflux). The basic kinetic properties of these three transport modes were analyzed. The kinetic properties closely resemble those of the reconstituted PIC from beef heart mitochondria. A competitive inhibitor of phosphate transport by the PIC, phosphonoformic acid, was used to establish functional overlap between the the physiological transport modes and the induced efflux mode. Replacement mutants were used to relate the reversible switch from antiport to uniport to a specific residue of the carrier. There are only three cysteines in the yeast PIC. They are at positions 28, 134, and 300 and were replaced by serine, both individually and in combinations. Cysteine 300 near the C-terminal loop and cysteine 134 located within the third transmembrane segment are accessible to bulky hydrophilic reagents from the cytosolic side, whereas cysteine 28 within the first transmembrane segment is not. None of the three cysteines is relevant to the two antiport modes. Cysteine 134 was identified to be the major target of bulky SH reagents, that lead to complete inactivation of the physiological transport modes. The reversible conversion between coupled antiport and uncoupled uniport of the PIC depends on the presence of one single cysteine (cysteine 28) in the PIC monomer, i.e. two cysteines in the functionally active dimer. The consequences of this result with respect to a functional model of the carrier protein are discussed.

Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa subunit with six transmembrane helices. The subunit possesses a sequence motif highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35 yeast (S. cerevisiae) MTP genes were resequenced since the masses of their proteins deviate significantly from the typical 30 kDa. We now find these four proteins to have 545, 285, 902, and 502 residues, respectively. Together with only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of some of the five residues that are absolutely conserved among the 12 MTPs with identified transport function and 17 other MTPs. We do now find these five consensus residues also in the new sequences of YNL083W and YFR045W. Additional analyses of the 35 yeast MTPs show that the location of transmembrane helix sequences do not correlate with the general consensus residues of the MTP family; protein segments connecting the six transmembrane helices and facing the intermembrane space are not uniformly short (about 20 residues) or long (about 40 residues) when facing the matrix; most MTPs have at least one transmembrane helix for which the sum of the negative hydropathy values of all residues yields a very small negative value, suggesting a membrane location bordering polar faces of other transmembrane helices or a non-transmembrane location. The extra residues of the three large MTPs are hydrophilic and at the N-terminal. The 200-residue N-terminal segment of YNL083W has four putative Ca2+-binding sites. The 500-residue N-terminal segment of YPR021C shows sequence similarity to enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is expressed solely during sporulation, while the expressions of YFR045W, YPR021C, and YDR470C are induced by various stress situations. These results also show that the 35 MTP genes are expressed under a rather diverse set of metabolic conditions that may help identify the function of the proteins. Interestingly, yeast two-hybrid screens, that will also be useful in identifying the function of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with non-MTPs.

The homodimeric mitochondrial phosphate transport protein (PTP), which has six transmembrane helices per subunit, catalyzes inorganic phosphate transport in an electroneutral and pH gradient-dependent manner across the inner membrane. We have replaced the Glu, Asp, and His residues of the yeast PTP to assess their role in the transport mechanism. Mutants with physiologically relevant transport activity were identified by their ability to rescue the PTP null mutant yeast from glycerol medium. Five residues appear critical for transport: His-32 in helix A, Glu-126 and -137 in helix C, and Asp-39 and -236 at the matrix ends of helices A and E. These mutant PTPs are expressed at near normal levels in yeast. This yeast PTP and the mutants were expressed in Escherichia coli as inclusion bodies, solubilized, purified, and reconstituted. Their transport activities correlate well with the physiological assays. None of the transport inactivating mutations appear to be due to major protein conformation changes as assayed by the efficiency of PTP incorporation into liposomes. Only the Glu95Gln (cytosolic helices B and C-connecting segment), Glu163Gln and Glu164Gln (matrix helices C and D-connecting segment), and Glu126Asp (helix C) show a near 70% decrease in liposome incorporation efficiency. In addition, mutations at either end of helix D increase phosphate transport 2-fold. We would like to suggest that Glu-126, His-32, and Glu-137 (similar to Asp-96, Lys-216, and Asp-85 of bacteriorhodopsin) form a proton cotransport pathway that is coupled in an as yet undefined manner (possibly via His-32) to a phosphate transport pathway, which may include helix D.

We have cloned the gene of the Saccharomyces cerevisiae phosphate transport protein (PTP), a member of the mitochondrial anion transport protein gene family. As PTP has a blocked N-terminus, we prepared three peptides. Oligonucleotides, based on their sequences, were used to screen a Yep24-housed genomic library. A total of 2073 bases of clone Y22 code for a 311 amino acid protein (Mr 32,814), which has similarities to the anion transport proteins: a triplicate gene structure and 6 hydrophobic segments. Typical for PTP, the triplicate gene structure possesses the X-Pro-X-(Asp/Glu)-X-X-(Lys/Arg)-X-(Arg/Lys)-X (X is an unspecified amino acid) motif and the very high homology only between the first and second repeat. The 6 hydrophobic segments harbor most of the 116 amino acids that are conserved between the yeast and the beef proteins. An N-terminal-extended signal sequence, as found in the beef protein, is absent. The yeast protein has about 33% fewer basic and acidic amino acids and five fewer Cys residues than the beef protein. The protein is insensitive to N-ethylmaleimide since Cys-42 (beef) has been replaced with a Thr. Mersalyl sensitivity has been retained and must be due to one of its three cysteines. Among these three cysteines, only Cys-28, located in the first hydrophobic segment, is conserved between the yeast and the beef protein.

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Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies. From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded. This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C 12 E 8 and dialysis. Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo-and heterodimeric forms of PIC, which are all fully active after reconstitution. As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer. We investigated its dimeric nature in the functionally active state after reconstitution. When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence. Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport. In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier. These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers.