Effects of mutations in the N terminal region of the yeast G protein alpha-subunit Gpa1p on signaling by pheromone receptors.

The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined. The C-terminus of the Galpha subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of Galpha as well. We screened for mutations in the N-terminal region of the Galpha subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast alpha-mating factor receptor. The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones alpha-factor and a-factor without affecting cellular levels of the subunit or its ability to interact with beta and gamma subunits. The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state. The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast Galpha subunit does not undergo extensive interactions with the alpha-factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.

Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis.

To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.

A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor.

Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)¿Ala(3)alpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.

Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae.

The alpha-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the alpha-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein alpha subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product.

Assembly of G protein-coupled receptors from fragments: identification of functional receptors with discontinuities in each of the loops connecting transmembrane segments.

The alpha-factor receptor of the yeast Saccharomyces cerevisiae is a member of the superfamily of G protein-coupled receptors that mediate signal transduction in response to sensory and chemical stimuli. All members of this superfamily contain seven predicted transmembrane segments. We have created a series of genes encoding alpha-factor receptors with amino- or carboxyl-terminal truncations at each of the loop regions connecting transmembrane segments. Split receptors containing a discontinuity in the peptide backbone were synthesized by coexpressing pairs of truncated receptor fragments in yeast. Complementary pairs of fragments split at sites within each of the cytoplasmic and extracellular loops were capable of assembling and transducing a signal in response to alpha-factor binding. One pair of noncomplementary fragments containing a deletion in the second intracellular loop of the receptor also yielded a functional receptor. Coexpression of certain combinations of overlapping fragments containing supernumerary transmembrane segments also led to formation of functional receptors, apparently because of proteolytic trimming of overlapping regions. Coexpression of truncated receptor fragments with full-length receptors had no effect on signaling by the full-length receptors. These results demonstrate the following: (1) Correct folding of the alpha-factor receptor does not require a covalent connection between any pair of transmembrane segments that are adjacent in the sequence. (2) Most of the second intracellular loop of the receptor is not required for function. (3) The structure of the receptor cannot, in most cases, tolerate the presence of extra transmembrane segments. (4) None of the truncated fragments of the alpha-factor receptor can efficiently oligomerize with normal receptors in such a way as to inhibit receptor function.

ATP-dependent transport of reduced glutathione on YCF1, the yeast orthologue of mammalian multidrug resistance associated proteins.

The transport systems involved in the export of cellular reduced glutathione (GSH) have not been identified, although recent studies implicate a role for some of the multidrug resistance associated proteins (MRP), including MRP1 and MRP2. The present study examined the hypothesis that the yeast orthologue of MRP, Ycf1p, mediates ATP-dependent GSH transport. [3H]GSH transport was measured in vacuolar membrane vesicles isolated from a control strain of Saccharomyces cerevisiae (DTY165), the isogenic DTY167 strain that lacks a functional Ycf1p, and in DTY167 transformed with a 2-micrometer plasmid vector containing YCF1. GSH transport in control vacuolar membrane vesicles was mediated largely by an ATP-dependent, low affinity pathway (Km = 15 +/- 4 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast Ycf1p transporter and inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, probenecid, and sulfinpyrazone, inhibitors of MRP1 and MRP2, but was minimally affected by membrane potential or pH gradient uncouplers. In contrast, ATP-dependent GSH transport was not seen in vacuolar membrane vesicles isolated from the DTY167 yeast strain without a functional Ycf1p but was restored to near wild-type levels in the DTY167 strain transformed with YCF1 and expressing the vacuolar Ycf1p transporter. On the other hand, expression and functional activity of a bile acid transporter, Bat1p, and of the V-type ATPase were similar in all three yeast strains. These results provide direct evidence for ATP-dependent low affinity transport of GSH by the yeast Ycf1p transporter. Because of the structural and functional homology between Ycf1p and MRP1 and MRP2, these data support the hypothesis that GSH efflux from mammalian cells is mediated by these membrane proteins.

ATP-dependent transport of reduced glutathione in yeast secretory vesicles.

Turnover of cellular reduced glutathione (GSH) is accomplished predominantly by export into the extracellular space; however, the plasma membrane transport mechanisms that mediate GSH efflux are not well characterized. The present study examined GSH transport using secretory vesicles isolated from the sec6-4 mutant strain of Saccharomyces cerevisiae. In contrast with studies in mammalian membrane vesicles, GSH transport in yeast secretory vesicles was mediated largely by an ATP-dependent, low-affinity pathway (Km 19+/-5 mM). ATP-dependent [3H]GSH transport was cis-inhibited by substrates of the yeast YCF1 transporter, including sulphobromophthalein, glutathione S-conjugates and the alkaloid verapamil, and was competitively inhibited by S-(2, 4-dinitrophenyl)glutathione (DNP-SG). Similarly, GSH competitively inhibited ATP-dependent [3H]DNP-SG transport, with a Ki of 18+/-2 mM, but had no effect on ATP-dependent [3H]taurocholate transport. ATP-dependent GSH transport was not affected by either membrane potential or pH-gradient uncouplers, but was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonate, probenecid and sulphinpyrazone, which are inhibitors of mrp1 and mrp2, mammalian homologues of the yeast YCF1 transporter. Western blot analysis of the secretory vesicle membrane fraction confirmed the presence of Ycf1p. These results provide the first direct evidence for low-affinity, ATP-dependent transport of GSH, and demonstrate that this ATP-dependent pathway displays kinetic characteristics similar to those of the yeast YCF1 transporter.

Bacterial expression of a mitochondrial cytochrome c. Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition.

Saccharomyces cerevisiae iso-1-cytochrome c has been expressed in Escherichia coli by coexpression of the genes encoding the cytochrome (CYC1) and yeast cytochrome c heme lyase (CYC3). Construction of this expression system involved cloning the two genes in parallel into the vector pUC18 to give the plasmid pBPCYC1(wt)/3. Transcription was directed by two promoters, Lac and Trc, that were located upstream from CYC1. Both proteins were expressed in the cytoplasm of E. coli cells harboring the plasmid. Semianaerobic cultures grown in a fermentor produced 15 mg of recombinant iso-1-cytochrome c per liter of culture. Attempts to increase production by addition of IPTG suppressed the number of copies of the CYC1 gene within the population. Wild-type iso-1-cytochrome c expressed with pBPCYC1(wt)/3 in E. coli was compared to the same protein expressed in yeast. At neutral pH, the two proteins exhibit indistinguishable spectroscopic and physical (Tm, Em') characteristics. However, electrospray mass spectrometry revealed that the lysyl residue at position 72 is not trimethylated by E. coli as it is by S. cerevisiae. Interestingly, the pKa of the alkaline transition of the protein expressed in E. coli is approximately 0.6 pKa unit lower than that observed for the cytochrome expressed in yeast (8.5-8.7). 1H NMR spectroscopy of the bacterially expressed cytochrome collected at high pH revealed the presence of a third alkaline conformer that is not observed in the corresponding spectrum of the cytochrome expressed in yeast. These observations suggest that Lys72 can serve as an axial ligand to the heme iron of alkaline iso-1-ferricytochrome c if it is not modified posttranscriptionally to trimethyllysine.

Genetic interactions among the transmembrane segments of the G protein coupled receptor encoded by the yeast STE2 gene.

G protein coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a wide variety of extracellular signals. However, the structural basis for activation of this class of receptors by ligand binding is not well understood. We report here the use of a systematic genetic protocol for identifying interactions among the seven transmembrane helices of the GPCR responsible for cellular responses to the alpha-mating pheromone of the yeast Saccharomyces cerevisiae. Random mutations were introduced into the region of the STE2 gene encoding the third transmembrane segment of the alpha-factor receptor, followed by screening for loss of signaling. The limited spectrum of non-conservative mutations recovered, including removal of the only negatively charged side-chain in the transmembrane region, indicates that most substitutions in the third transmembrane segment do not affect receptor function. Three second-site intragenic suppressors of these initial mutations were isolated following mutagenesis of the remaining six transmembrane segments. One of these suppressors, Y266C in the sixth transmembrane segment, is allele specific and shows non-additivity of phenotypes indicative of a physical interaction between the third and sixth transmembrane regions of the receptor. A second suppressor, M218T in the fifth transmembrane segment, exhibits only partial allele specificity. A third suppressor, R58G, in the first transmembrane segment, suppresses a variety of starting alleles and appears to cause global stabilization of the receptor. Analysis of these suppressors and additional alleles can provide a database for modeling GPCR structure.

Sequence requirements for mitochondrial import of yeast cytochrome c.

Apocytochrome c is synthesized in the cytoplasm, transported to the mitochondrial intermembrane space, and subsequently covalently attached to heme in a reaction catalyzed by the enzyme cytochrome c heme lyase. We have investigated the amino acid sequences in cytochrome c which are required for mitochondrial import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. Import of the altered apocytochromes c was assayed in yeast strains that overexpressed cytochrome c heme lyase. Under these conditions, there was efficient mitochondrial accumulation of forms of apocytochrome c which are incapable of having heme covalently attached. In fact, all apocytochromes c containing deletions located to the carboxyl-terminal side of His27 efficiently accumulated in the mitochondria of strains overexpressing heme lyase, even though all but one of these deletion-containing proteins were incapable of heme attachment. A minimum length of polypeptide chain at the extreme amino terminus of cytochrome c, rather than any specific sequence element in this region, appears to be required for efficient mitochondrial import. Certain amino acid substitutions in the region extending from Gly15 to Leu18, at residue Phe19 and at residue His27, lead to reduced mitochondrial import of apocytochrome c, resulting from stalling of the altered apocytochrome c in partially imported states.

Noncovalent binding of heme induces a compact apocytochrome c structure.

Mitochondrial holocytochrome c contains heme that is covalently attached to the protein in a reaction catalyzed by the enzyme cytochrome c heme lyase. In the absence of heme, apocytochrome c, the precursor to holocytochrome c, is unfolded. We find that purified apocytochrome c binds noncovalently to heme. Binding is accompanied by changes in the optical absorption spectrum of heme and by quenching of the tryptophan fluorescence of the protein. The affinity of apocytochrome c for heme, as well as the stoichiometry of binding, appears to depend on whether or not cyanide is present and on the oxidation state of heme. Under reducing conditions, in the presence of cyanide, the association appears to be 1:1, with a binding constant of about 10(7) M-1. Under oxidized conditions, there may be multiple hemes bound per molecule of apocytochrome c. Upon binding to heme, apocytochrome c exhibits a mobility similar to that of holocytochrome c in gel filtration chromatography and velocity gradient ultracentrifugation, indicating that the heme-protein complex adopts a structure that is almost as compact as that of holocytochrome c. Changes in the circular dichroism spectrum of apocytochrome c are consistent with an increase in the alpha-helical content of the protein on binding heme. The compact structure of the noncovalent heme-apocytochrome c complex may represent an intermediate in the de novo folding of cytochrome c.

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.

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

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.

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c.

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.

The pH-dependent conformational change of diphtheria toxin.

Labeling by a hydrophobic photoactivatable reagent and limited proteolysis have been used to study conformational changes of diphtheria toxin related to its pH-dependent membrane insertion and translocation. TID (3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine) labels diphtheria toxin at pH 5 much more efficiently than at pH 7, both in the presence and absence of lipid vesicles. In the absence of membranes, the extent of labeling is greater and the pH dependence is stronger. As analyzed on sodium dodecyl sulfate-polyacrylamide gels and by high pressure liquid chromatography, both the A- and B-subunits and most of the cyanogen bromide fragments of the toxin are labeled by TID at acid pH. The products of trypsin cleavage of diphtheria toxin at pH 5 are different from those seen at neutral pH. Trypsin-susceptible sites were identified by gel electrophoresis of the trypsin fragments, combined with electrophoresis and high pressure liquid chromatography of CNBr digests of trypsin-treated toxin. At neutral pH, the main sites of digestion are at the junction between the A- and B-fragments and near the NH2 terminus of the A-fragment. At pH 5.2, these sites are less efficiently cut, and new sites appear near the NH2 terminus of the B-fragment, in an amphipathic portion of the sequence. Thus, even in the absence of membranes, acid pH induces a significant conformational change in diphtheria toxin. This change involves burial of some previously accessible sites, exposure of previously inaccessible sites, and the formation of hydrophobic regions over an extensive portion of the polypeptide chain.

[Histopathology of the liver and kidney during malaria: relation to malaria-induced dyslipoproteinemia]. / Histopathologie du foie et du rein au cours du paludisme: rapports avec la dyslipoprotéinémie palustre.

A kinetic study concerning histologic and cytologic alterations during Plasmodium chabaudi infection of Swiss mice has been carried out. In liver, a reversible focal and non ischemic necrosis and a vascular congestion were observed together with an accumulation of malarial pigment. The endoplasmic reticulum cisternae and Golgi saccules of hepatocytes were highly distended. Hepatocyte microvilli in biliary canaliculi and in Disse' spaces were markedly less developed and less numerous than in normal liver. Intracytoplasmic lipid globules were found in large amount in hepatocytes before the peak of parasitaemia. Their number and size gradually diminished thereafter. Hepatocytic mitochondria showed important unspecific modifications probably in relation, at last partly, to the tissue anoxia. Some hepatocytic changes (intracytoplasmic lipid globules, enlargement of endoplasmic reticulum cisternae and Golgi saccules) were consistent with an increased synthesis of lipoproteins (VLDL). The kidney showed only minor histological and ultrastructural changes. However haemosiderin was observed in proximal tubules and in their bordering cells. The deposit of immune complex reported previously do not appear associated with tissular or cellular important alterations.

Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae.

Mitochondrial cytochrome c contains a heme group covalently attached through thioether linkages to two cysteinyl residues of the protein. We demonstrate here that the nuclear gene, CYC3, in the yeast Saccharomyces cerevisiae, encodes cytochrome c heme lyase (CCHL), the enzyme catalyzing the attachment of heme to apocytochrome c. Mitochondrial extracts from cyc3- mutants are deficient in CCHL activity compared with extracts from normal strains, whereas strains carrying multiple copies of the CYC3 gene exhibit high levels of the activity. The CYC3 gene was cloned by functional complementation of a cyc3- mutant using a previously isolated plasmid containing the gene PYK1, which is tightly linked to CYC3. An open reading frame encoding a protein of 269 amino acids was identified from the DNA sequence of a fragment encompassing the CYC3 gene, and the corresponding transcript shown to be approximately 0.9 kb in length. CCHL appears to be a single polypeptide chain which acts specifically on the two forms of cytochrome c, but not on cytochrome c1.

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis.

Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated 3H leucine from the digested membranes. During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.

Insertion of apocytochrome c into lipid vesicles.

Apocytochrome c (cytochrome c without the heme) is synthesized in the cell cytoplasm without a cleaved signal sequence, then transported across the outer mitochondrial membrane. We have studied the interaction of apocytochrome c with lipid vesicles as a model for understanding protein translocation across membranes. Apocytochrome c (but not holocytochrome c) that has been incubated with vesicles at 37 degrees C in 0.2 M NaCl binds to the vesicles. Under these conditions, as well as upon incubation with detergent or at high protein concentrations, all the added protein remains partly accessible to externally added protease, but a COOH-terminal fragment of some of the protein molecules becomes protected against digestion. When apocytochrome c is added to azolectin vesicles with internally trapped proteases, most of the added protein can be digested, even in the presence of a large excess of protease inhibitor external to the vesicles. Thus, in spite of a lack of nonpolar stretches in its amino acid sequence, apocytochrome c is capable of binding to and inserting into lipid membranes. In this model system, transport may be driven by trapping of protease-digested apocytochrome c on one side of the membrane.