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Background: Historically, the treatment of iliac artery occlusive disease required a surgical bypass usually consisting of an aortobifemoral bypass or an iliofemoral bypass. With the advent of balloon angioplasty and stenting, these procedures are frequently replaced with endovascular options. However, the treatment of diffuse occlusive disease of the external iliac artery (EIA) using balloon angioplasty and/or stenting does not carry a favorable long-term patency rate. Remote endarterectomy of the EIA using ring dissectors with balloon assistance provides a novel, controlled, safe, and durable treatment of the diseased and/or occluded EIA., Methods: A retrospective review over the past 6 years was performed at our institution identifying patients treated with balloon-assisted remote endarterectomy of the EIA by the current five practicing vascular surgeons. The technique involves exposure of the ipsilateral common femoral artery. With nonocclusive disease, direct access into the common femoral artery is performed, a wire is traversed through the diseased EIA, and a balloon is inflated at the origin of the vessel providing hemostasis and control. A femoral endarterectomy is performed, and a ring dissector is passed over the endarterectomized material including the wire and balloon catheter and advanced remotely through the EIA up to the balloon. The balloon is briefly deflated, repositioned within the ring dissector, and reinflated, thus cutting the plaque. This allows for retraction of the inflated balloon and cutter, removing the endarterectomized core plaque. The procedure is similar for the treatment of an occluded EIA, but wire access across the occluded vessel is normally achieved with contralateral access. In both cases, the balloon provides control and hemostasis and is critically important in the rare treatment of vessel rupture., Results: A total of 101 vessels were treated in 97 patients. The procedure was successful in 98 vessels (97%) with failure related to vessel rupture requiring conversion to an iliofemoral bypass. The estimated patency rate at three years was 94% with a median follow-up of 20 months. Restenosis/occlusion in four patients seemed to be related to a severe sclerotic response. The EIA was occluded 32% of the time. The common iliac artery (CIA) was diseased requiring angioplasty and stenting 29% of the time and a stent was placed at the transition zone between endarterectomized vessel and nontreated proximal most EIA or distal most CIA 58% of the time. There were no perioperative deaths., Conclusions: Balloon-assisted remote endarterectomy of the diffusely diseased and/or occluded EIA is a safe and durable option. It precludes the need for a prosthetic conduit and the risk of associated infection. It also involves a single groin incision and negates the need for retroperitoneal exposure of the CIA., (Copyright © 2019 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

The emergence of highly pathogenic human coronaviruses (hCoVs) in the last two decades has illuminated their potential to cause high morbidity and mortality in human populations and disrupt global economies. Global pandemic concerns stem from their high mortality rates, capacity for human-to-human spread by respiratory transmission, and complete lack of approved therapeutic countermeasures. Limiting disease may require the development of virus-directed and host-directed therapeutic strategies due to the acute etiology of hCoV infections. Therefore, understanding how hCoV-host interactions cause pathogenic outcomes relies upon mammalian models that closely recapitulate the pathogenesis of hCoVs in humans. Pragmatism has largely been the driving force underpinning mice as highly effective mammalian models for elucidating hCoV-host interactions that govern pathogenesis. Notably, tractable mouse genetics combined with hCoV reverse genetic systems has afforded the concomitant manipulation of virus and host genetics to evaluate virus-host interaction networks in disease. In addition to assessing etiologies of known hCoVs, mouse models have clinically predictive value as tools to appraise potential disease phenotypes associated with pre-emergent CoVs. Knowledge of CoV pathogenic potential before it crosses the species barrier into the human population provides a highly desirable preclinical platform for addressing global pathogen preparedness, an overarching directive of the World Health Organization. Although we recognize that results obtained in robust mouse models require evaluation in non-human primates, we focus this review on the current state of hCoV mouse models, their use as tractable complex genetic organisms for untangling complex hCoV-host interactions, and as pathogenesis models for preclinical evaluation of novel therapeutic interventions.

The recurrence of new human cases of Middle East respiratory syndrome coronavirus (MERS-CoV) underscores the need for effective therapeutic countermeasures. Nonhuman primate models are considered the gold standard for preclinical evaluation of therapeutic countermeasures. However, MERS-CoV-induced severe respiratory disease in humans is associated with high viral loads in the lower respiratory tract, which may be difficult to achieve in nonhuman primate models. Considering this limitation, we wanted to ascertain the effectiveness of using a MERS-CoV infectious clone (icMERS-0) previously shown to replicate to higher titers than the wild-type EMC 2012 strain. We observed respiratory disease resulting from exposure to the icMERS-0 strain as measured by CT in rhesus monkeys with concomitant detection of virus antigen by immunohistochemistry. Overall, respiratory disease was mild and transient, resolving by day 30 post-infection. Although pulmonary disease was mild, these results demonstrate for the first time the utility of CT imaging to measure disease elicited by a MERS-CoV infectious clone system in nonhuman primate models.

We recently established a mouse model (288-330 +/+ ) that developed acute respiratory disease resembling human pathology following infection with a high dose (5 × 10 6 PFU) of mouse-adapted MERS-CoV (icMERSma1). Although this high dose conferred fatal respiratory disease in mice, achieving similar pathology at lower viral doses may more closely reflect naturally acquired infections. Through continued adaptive evolution of icMERSma1 we generated a novel mouse-adapted MERS-CoV (maM35c4) capable of achieving severe respiratory disease at doses between 10 3 and 10 5 PFU. Novel mutations were identified in the maM35c4 genome that may be responsible for eliciting etiologies of acute respiratory distress syndrome at 10-1000 fold lower viral doses. Importantly, comparative genetics of the two mouse-adapted MERS strains allowed us to identify specific mutations that remained fixed through an additional 20 cycles of adaptive evolution. Our data indicate that the extent of MERS-CoV adaptation determines the minimal infectious dose required to achieve severe respiratory disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward. IMPORTANCE The initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants., (Copyright © 2017 Menachery et al.)

The onset and progression of cancer is associated with the methylation-dependent silencing of specific genes, however, the mechanism and its regulation have not been established. We previously demonstrated that reduction of mitochondrial DNA content induces cancer progression. Here we found that mitochondrial DNA-deficient LNrho0-8 activates the hypermethylation of the nuclear DNA promoters including the promoter CpG islands of the endothelin B receptor, O6-methylguanine-DNA methyltransferase, and E-cadherin. These are unmethylated and the corresponding gene products are expressed in the parental LNCaP containing mitochondrial DNA. The absence of mitochondrial DNA induced DNA methyltransferase 1 expression which was responsible for the methylation patterns observed. Inhibition of DNA methyltransferase eliminated hypermethylation and expressed gene products in LNrho0-8. These studies demonstrate loss or reduction of mitochondrial DNA resulted in the induction of DNA methyltransferase 1, hypermethylation of the promoters of endothelin B receptor, O6-methylguanine-DNA methyltransferase, and E-cadherin, and reduction of the corresponding gene products.

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

To better define the regulatory role of the F(1)-ATPase alpha-subunit in the catalytic cycle of the ATP synthase complex, we isolated suppressors of mutations occurring in ATP1, the gene for the alpha-subunit in Saccharomyces cerevisiae. First, two atp1 mutations (atp1-1 and atp1-2) were characterized that prevent the growth of yeast on non-fermentable carbon sources. Both mutants contained full-length F(1)alpha-subunit proteins in mitochondria, but in lower amounts than that in the parental strain. Both mutants exhibited barely measurable F(1)-ATPase activity. The primary mutations in atp1-1 and atp1-2 were identified as Thr(383) --> Ile and Gly(291) --> Asp, respectively. From recent structural data, position 383 lies within the catalytic site. Position 291 is located near the region affecting subunit-subunit interaction with the F(1)beta-subunit. An unlinked suppressor gene, ASC1 (alpha-subunit complementing) of the atp1-2 mutation (Gly(291) --> Asp) restored the growth defect phenotype on glycerol, but did not suppress either atp1-1 or the deletion mutant Deltaatp1. Sequence analysis revealed that ASC1 was allelic with RAS2, a G-protein growth regulator. The introduction of ASC1/RAS2 into the atp1-2 mutant increased the F(1)-ATPase enzyme activity in this mutant when the transformant was grown on glycerol. The possible mechanisms of ASC1/RAS2 suppression of atp1-2 are discussed; we suggest that RAS2 is part of the regulatory circuit involved in the control of F(1)-ATPase subunit levels in mitochondria.

The authors believe that the aging process--the loss of youthful resilience--is caused by the decline of many hormones. By restoring these hormone levels, the decay associated with old age can be eliminated and in some cases, perhaps reversed. The hormones that naturally occur in the body should be replenished through a medically sound regimen. The hormones must work together. The data indicate that aging occurs at two levels: systemically and cellularly. Systemic controls regulate the rates of intracellular enzymatic processes that accelerate to protect against aging.

Background: Ultrasonography-guided compression repair is reported to be effective therapy for femoral pseudoaneurysms that develop after catheterization procedures. This study summarizes our experience with color-flow duplex-guided repair of these lesions., Methods: A retrospective chart review of all patients who underwent this procedure was undertaken, with statistical analysis to identify factors associated with success., Results: Compression repair of 69 pseudoaneurysms was attempted. Pseudoaneurysms developed after therapeutic catheterization in 48 patients and after diagnostic procedures in 21. Sites of arterial puncture were the common femoral artery in 59 patients and the superficial femoral or profunda femoris arteries in 10. Diameters of the pseudoaneurysms ranged from 3 to 60 mm (mean, 28 mm). Compression was attempted at a mean of 5 days (range, 1 to 21 days) after catheterization. Compression produced complete thrombosis of the pseudoaneurysm at the initial attempt in 43 (62%) of 69 patients. With repeated attempts the ultimate success was 47 (68%) of 69. Success was achieved in 44 (75%) of 59 common femoral pseudoaneurysms but in only 3 (30%) of 10 superficial femoral or profunda femoris lesions (p = 0.009). Anticoagulation, sheath size, pseudoaneurysm chamber size, and time between catheterization and compression were not significantly different between lesions that were successfully compressed and those that were not. No ischemic or embolic complications were observed., Conclusions: Color-flow duplex-guided compression repair can be safely attempted as the initial therapy for all uncomplicated pseudoaneurysms arising from the common femoral artery after catheterization, with the expectation of success in most.

Most proteins synthesized in cytoplasm target to mitochondria through sequences at their amino termini. However, a previous study suggests that the native carboxyl terminus of ISP6 might be critical of its specific delivery. Here we investigated the sequence directing ISP6 to yeast mitochondrial outer membrane. Unlike mitochondrial presequences, a region at the amino terminus of ISP6 is dispensable for importing the rest of the protein. The carboxyl-terminal end and the nearby transmembrane region of ISP6 are essential to direct the protein exclusively to its correct membrane destination. ISP6 thus may be directed to mitochondria by an unusual sequence.

The 46-kDa protein YDJ1 is one of several known yeast homologues of the Escherichia coli DnaJ protein. Like all J homologues, it shares homology with the highly conserved NH2-terminal "J-domain" of DnaJ. A component of the DnaK (Hsp70) chaperone machinery that mediates protein folding, DnaJ is necessary for survival at elevated temperatures. It stimulates ATP hydrolysis by DnaK and effects the release of DnaK-bound polypeptides. Previous genetic and biochemical studies indicate that the J-domain is necessary for these functions. Using peptides corresponding to J-domain sequence, we show that a peptide containing the highly conserved His-Pro-Asp sequence at positions 34-36 in the J-domain competes off YDJ1 stimulation of Hsp70 ATPase activity. Inhibitory concentrations of peptide do not prevent binding of folding substrates, therefore YDJ1 must interact with Hsp70 at a site distinct from that for substrate binding. This interaction is critical for Hsp70 activity, since a mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates neither Hsp70 ATPase nor substrate release. The importance of the proper function of this region of the protein is supported by the poor growth and temperature-sensitive phenotype of yeast expressing ydj1Q34.

Improve resolution of computed tomography (CT) and ultrasonography allows us to visualize the proximal extent of biliary obstruction and the presence of a periampullary mass in most patients with malignant extrahepatic biliary obstruction. Our purpose in this report is to challenge the need for preoperative percutaneous biopsy, endoscopic retrograde cholangiopancreatography, or preoperative placement of a biliary endoprosthesis in the good-risk patient in whom the imaging procedure clearly defines a periampullary mass and the proximal extent (hepatic extent) of biliary obstruction. We recently managed three patients in whom one of these invasive procedures led to a complication that delayed, prevented, or complicated appropriate operative resection of a pancreatic neoplasm. Because a negative percutaneous biopsy, cholangiographic imaging of a dilated bile/pancreatic duct clearly seen on CT or ultrasonography, or short-term preoperative biliary decompression does not alter the decision for operative exploration and may cause complications, we argue against their use in the good-risk patient with both extrahepatic biliary obstruction and a periampullary pancreatic mass well delineated on noninvasive imaging.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.

To study the biogenesis of ISP6, an outer membrane component of the mitochondrial protein translocation complex, two fusion proteins have been made by fusing ISP6 to either the carboxyl- or amino-terminal end of the mouse dihydrofolate reductase (DHFR). In vitro import experiments showed that when DHFR was placed at the carboxyl-terminal end of ISP6, the resulting fusion protein 6-DHFR inserted into mitochondrial membrane less efficiently than the other form of the fusion proteins. In vivo this fusion protein lost its ability to suppress the temperature-sensitive phenotype of an isp42 mutant, while the other fusion protein DHFR-6, which was found targeted correctly to mitochondria, suppressed the mutant as well as the wild-type ISP6. Further analysis showed that the binding and insertion of DHFR-6 to mitochondrial outer membrane was not affected by deletion of either of the two mitochondrial protein receptors or by the predigestion of mitochondrial surface proteins prior to import. Additional data indicated that ISP42, which closely associates with ISP6 in the translocation complex, does not likely play the role of a targeting partner for ISP6. In summary, these data suggest that ISP6 may target to mitochondria by sequences at its carboxyl terminus and that the import process of ISP6 is most likely distinct from that of most other mitochondrial precursors, which are recognized by protein receptors on mitochondrial surface.

In Saccharomyces cerevisiae Ydj1p, a DnaJ homolog, is localized to the cytosol with the Ssa and Ssb Hsp70 proteins. Ydj1p helps facilitate polypeptide translocation across mitochondrial and endoplasmic reticulum membranes (Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1992) Cell 71, 1143-1155) and can directly interact with Ssa1p to regulate chaperone activity (Cyr, D. M., Lu, X., and Douglas, M. G. (1992) J. Biol. Chem. 267, 20927-20931). In this study, the role of Ydj1p in modulating ATP-dependent reactions catalyzed by Ssa and Ssb Hsp70 proteins has been examined using purified components and compared with that of other Hsp70 homologs BiP and DnaK. Ssa1p, Ssa2p, and Ssb1/2p all formed stable complexes with the mitochondrial presequence peptide, F1 beta(1-51). ATP alone had only modest effects on polypeptide complex formation with Ssa1p and Ssa2p, but prevented the majority of polypeptide binding to BiP and DnaK. ATP by itself also reduced polypeptide binding to Ssb1/2p to a level that was intermediate between that observed for the Ssa Hsp70 proteins tested and BiP and DnaK. ATP hydrolysis by Ssa1p, Ssa2p, and Ssb1/2p occurred at similar rates. Ydj1p was a potent modulator of the both the ATPase and polypeptide binding activities of Ssa1p and Ssa2p. In contrast, Ydj1p had little effect on the ATPase and polypeptide binding activity of Ssb1/2p. Therefore the chaperone-related activities of Ssa and Ssb Hsp70 proteins exhibit significant differences in sensitivity to ATP and YDJ1p. These data indicate that regulation of Hsp70 activity by DnaJ homologs can be specific. The specificity of interactions between Ydj1p and the Ssa and Ssb Hsp70 proteins observed could contribute in determining the functional specificity of these chaperones in the cytosol. In related experiments, F1 beta(1-51) was found to reduce the extent to which Ydj1p stimulated Ssa1p ATPase activity. This effect correlated with the formation of F1 beta(1-51).Ssa1p complexes. We propose that intramolecular communication between the polypeptide binding, ATPase and DnaJ regulatory domains on Ssa1p plays a role in the regulation of chaperone activity.

The folding of proteins and the assembly of protein complexes within subcompartments of the eukaryotic cell is catalysed by different members of the Hsp70 protein family. The chaperone function of Hsp70 proteins in these events is regulated by members of the DnaJ-like protein family, which occurs through direct interaction of different Hsp70 and DnaJ-like protein pairs that appear to be specifically adapted to each other. This review highlights the diversity of functions of DnaJ-like proteins by using specific examples of DnaJ-Hsp70 interactions with polypeptides in yeast protein-biogenesis pathways.

To search genetically for additional components of the protein translocation apparatus of mitochondria, we have used low fidelity PCR mutagenesis to generate temperature-sensitive mutants in the outer membrane translocation pore component ISP42. A high copy number suppressor of temperature-sensitive isp42 has been isolated and sequenced. This novel gene, denoted ISP6, encodes a 61 amino acid integral membrane protein of the mitochondrial outer membrane, which is oriented with its amino-terminus facing the cytosol. Disruption of the ISP6 gene is without apparent effect in wild type yeast cells, but is lethal in temperature-sensitive isp42 mutants. Immunoprecipitation of the gene product, ISP42p, from mitochondria solubilized under mild conditions reveals a multi-protein complex containing ISP6p and ISP42p.

Structure-function relationships in the yeast adenine nucleotide carrier (AAC2) were probed by genetic selection techniques in Saccharomyces cerevisiae. As described in the preceding paper, yeast require a functional AAC2 (or AAC1) to grow on a non-fermentable carbon source. Mutants of AAC2 that could not grow on glycerol were subjected to selection for spontaneous suppressors. Ile mutants in the 100% conserved matrix Arg triplet R252 to R254 proved amenable to this approach, yielding colonies on glycerol plates at modest frequency. All mutants analyzed were single point mutations within the AAC2 gene at a different site than the Arg cluster. R254I gave the largest number (11) of unique revertants, while R253I gave only four and R252I gave none, thus, there was a gradient of effect in mutations of the Arg cluster. Unexpectedly, 14 of the 15 revertant mutations affected 13 different amino acids in a narrow sector of the AAC2 topological map, near the cytosolic surface. These mutants are proposed to be in, or very near, the membrane on the opposite side from the matrix Arg cluster. Helical wheel projections of the transmembrane segments show, with one exception, that mutations and charged residues fall within half of each helix. These mutants appear either to line the throat of the membrane channel, or to be involved in helix contacts near the cytosolic face of the inner mitochondrial membrane. These suppressors place further limitations on the organization of the nucleotide channel. We present a model of the AAC2 nucleotide channel based on these results. The region defined by these suppressors is dynamically linked to the Arg cluster through the function of the AAC2 protein. Discrete structures defined by multiple revertant mutations will likely be a common feature of similar regain-of-function schemes, especially when applied to membrane transport proteins. We propose these functionally mapped regions of proteins be named morphological units or morphs for short.

The ADP/ATP translocator mediates adenine nucleotide exchange across the inner mitochondrial membrane. ADP/ATP exchange is essential when yeast are grown on a non-fermentable carbon source such as glycerol, but it is not required for growth on glucose. Failure to grow on glycerol is therefore a phenotypic indicator of protein function, and it has been used here to screen site-directed mutants to identify functionally important amino acids in the yeast adenine nucleotide translocator (AAC2). Single mutations of all four charged amino acids in the transmembrane segments of AAC2 (K38A, R96D, R96H, R96L, R96P, R204L, R294A) resulted in loss of function, as did mutations in the matrix arginine cluster (R252I, R253I, R254I). Seven other residues were mutated without affecting growth on glycerol (C73S, C244S, C271S, K179M, K182I, P247G, W235F). The non-functional mutants have been used to select intragenic suppressors to gain further insight into the structure of this membrane transport protein.

An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomyces cerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as well as activities involving chromosomal DNA pairing and mechanics. Preliminary analysis of an hke1ts mutant reveals a precipitous decline in the translation of mRNA at the nonpermissive temperature. Sporulation of heterozygous HKE1/hke1::URA3 diploids reveals that this gene, unlike the highly related XRN1/KEM1 gene, is essential for cell viability. Overexpression of the homologous gene product, p175, failed to rescue cells lacking a functional p116. In vitro studies demonstrate that p116 is a protein with 5'-->3' exoribonuclease activity, a major activity of the related p175. An immunoreactive RNase activity of 116 kDa is abolished with antiserum against p116. Both the level of this protein and the RNase activity correlate with HKE1 gene dosage. The RNase activity purifies coincidentally with a previously described 116-kDa RNase having 5'-->3' exoribonuclease activity.

The role of S. cerevisiae YDJ1 protein (YDJ1p) in polypeptide translocation across membranes has been examined. A conditional ydj1 mutant strain (ydj1-151TS) is defective for import of several polypeptides into mitochondria and alpha factor into the endoplasmic reticulum at 37 degrees C. These defects are suppressed by E. coli dnaJ or overexpression of S. cerevisiae SIS1 proteins. A different ydj1 mutant, which cannot be farnesylated (ydj1-S406), displays similar transport defects to the ydj1-151 strain. Furthermore, the ability of purified ydj1-151p to stimulate the ATPase activity of hsp70SSA1 was greatly diminished compared with the wild-type protein. Together, these data suggest that YDJ1p functions in polypeptide translocation in a conserved manner, probably acting at organelle membranes and in association with hsp70 proteins.

We report that a purified cytoplasmic Hsp70 homolog from Saccharomyces cerevisiae, Hsp70SSA1, exhibits a weak ATPase activity, which is stimulated by a purified eukaryotic dnaJp homolog (YDJ1p). Stable complex formation between Hsp70SSA1 and the permanently unfolded protein carboxymethylated alpha-lactalbumin (CMLA) was assayed by native gel electrophoresis. The affinity of Hsp70SSA1 for CMLA appeared to be regulated by YDJ1p. Significant reduction in both CMLA-Hsp70SSA1 complex formation and the release of CMLA pre-bound to Hsp70SSA1 was observed only in the presence of both YDJ1p and ATP. Thus, Hsp70SSA1 and YDJ1p interact functionally in the execution of Hsp70SSA1 chaperone activities in the eukaryotic cell.

The Saccharomyces cerevisiae YDJ1 protein (YDJ1p) contains a C-terminal "CaaX box" motif common to proteins that are modified by prenylation. In the present study we show that YDJ1p is a specific substrate for both yeast and mammalian protein farnesyltransferase enzymes in vitro. A mutant form of YDJ1p, in which the conserved cysteine of the CaaX box is mutated to a serine (ydj1-S406p), cannot be farnesylated in vitro. After expression in S. cerevisiae, ydj1-S406p displays a reduced electrophoretic mobility and an increased cytosolic localization in subcellular fractionation experiments when compared to wild type YDJ1p. Expression of ydj1-S406 in cells lacking YDJ1 results in a temperature-sensitive growth phenotype in S. cerevisiae. These data indicate that farnesylation of YDJ1p is required for its function at elevated temperatures.

The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.

Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G. (1991) J. Biol. Chem. 266, 21693-21699). Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20. A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2. Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import. Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein. Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition. F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25%. In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25%. Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi. Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions. F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step. Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide. We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria.

The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus. Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane. To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry. These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure. Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior. Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer. High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors. Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site. These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.

The YDJ1 (yeast dnaJ) gene was isolated from a yeast expression library using antisera made against a yeast nuclear sub-fraction termed the matrix lamina pore complex. The predicted open reading frame displays a 32% identity with the sequence of the Escherichia coli heat shock protein dnaJ. Localization of YDJ1 protein (YDJ1p) by indirect immunofluorescence reveals it to be concentrated in a perinuclear ring as well as in the cytoplasm. YDJ1p cofractionates with nuclei and also microsomes, suggesting that its perinuclear localization reflects association with the ER. YDJ1p is required for normal growth and disruption of its gene results in very slow growing cells that have pleiotropic morphological defects. Haploid cells carrying the disrupted YDJ1 gene are inviable for growth in liquid media. We further show that a related yeast protein, SIS1, is a multicopy suppressor of YDJ1.

The respiratory deficient nuclear mutant of Saccharomyces cerevisiae, N9-168, assigned to complementation group G95 was previously shown to lack subunit 9, one of the three mitochondrially encoded subunits of the Fo component of the mitochondrial ATPase. As a consequence of the structural defect in Fo, the ATPase activity of G95 mutants is not inhibited by rutamycin. The absence of subunit 9 in N9-168 has been correlated with a lower steady-state level of its mRNA and an increase in higher molecular weight precursor transcripts. These results suggest that the mutation is most likely to affect either translation of the oli1 mRNA or processing of the primary transcript. We have isolated a nuclear gene, designated ATP13, which complements the respiratory defect and restores rutamycin-sensitive ATPase in G95 mutants. Disruption of ATP13 induces a respiratory deficiency which is not complemented by G95 mutants. The nucleotide sequence of ATP13 indicates a primary translation product with an Mapp of 42,897. The protein has a basic amino terminal signal sequence that is cleaved upon import into mitochondria. No significant primary structure homology is detected with any protein in the most recent libraries.

The gene encoding the major ADP/ATP carrier in yeast AAC2 (pet9; Lawson, J., and Douglas, M. (1988) J. Biol. Chem. 263, 14812-14818) has been disrupted (delta AAC2) by itself and in combination with a disruption of a second translocator gene AAC1 (delta AAC1). Disruption of AAC2 like the pet9 mutation renders yeast unable to grow on a nonfermentable carbon source. The AAC1 AAC2 double disruption exhibits a phenotype identical to the AAC2. This provides the host strain for the analysis of point mutations in the AAC protein. We have initiated this structure-function analysis by characterizing and confirming that the pet9 mutation is a G to A transition resulting in an arginine to histidine change at position 96. Site-directed replacements at Arg96 confirm its essential function for growth on a nonfermentable carbon source. These data also suggest that in the absence of functional AAC1 and AAC2 gene products, adenine nucleotide transport across the mitochondrial inner membrane must occur by an as yet unidentified translocator or translocation mechanism or that within these cells separate intra- and extramitochondrial adenine nucleotide pools can exist to support growth.

AAC1 and AAC2 genes in yeast each encode functional ADP/ATP carrier (AAC) proteins of the mitochondrial inner membrane. In the present study, mitochondria harboring distinct AAC proteins and the pet9 Arg96 to HIS mutant (Lawson, J., Gawaz, M., Klingenberg, M., and Douglas, M. G. (1990) J. Biol. Chem. 265, 14195-14201) protein have been characterized. In addition, properties of the different AAC proteins have been defined following reconstitution into proteoliposomes. Deletion of AAC2 but not AAC1 causes a major reduction in the mitochondrial cytochrome content and respiration, and this level remains low even when the level of AAC1 protein is increased to 20% that of the AAC2 gene product. In reconstitution studies, the rate of nucleotide transport by isolated AAC1 protein is approximately 40% that of the AAC2 protein. Thus, the lack of mitochondrial-dependent growth supported by the AAC1 gene product alone may be due to the combination of low abundance and reduced activity. Surprisingly, analysis of the Arg96 to His mutant protein revealed binding and transport activities similar to the functional AAC1 and AAC2 gene products. These observations are discussed in relation to a molecular analysis of this highly conserved small transporter and its function in conjunction with other proteins in the mitochondrial membrane.

Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coli is technically simple and yields transformants with an efficiency of 10(6)-10(7) transformants/microgram DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. coli reveals basic differences between the PEG and standard CaCl2 methods for transformation of E. coli. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.

Fractions enriched for nuclear pore complexes (NPCs) have been isolated from Saccharomyces cerevisiae. The sequential extraction of nuclei with detergent, nucleases, and salt reveals an organization of the yeast NPC similar to other eukaryotes. Yeast NPCs contain a 30-nm "ring" structure not previously described in other organisms. This structure appears to organize 10-nm filaments into an assembly which exhibits an eight-fold rotational symmetry. Some proteins in the NPC fraction are capable of forming intermediate-sized filaments. These studies suggest that some component of the nuclear pore complex organizes an interaction between nuclear and cytoplasmic networks of intermediate filaments.

A comparison was made of the subunit sensitivities of the F1-ATPase and the Triton-solubilized ATPase complex to trypsin degradation. The dissociation of the F1-ATPase from ATPase complex increased the trypsin sensitivity of subunit 3 by a factor of 2 and increased the sensitivity of a particular trypsin site (or group of sites) on subunit 1 by 7-fold. The overall degradation of subunits 1 and 2 appears to be the same in solubilized ATPase complex and the F1-ATPase. Implications of these findings for structural models of the ATPase complex are discussed.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.

The ADP/ATP carrier of yeast (309 amino acids) is an abundant transmembrane protein of the mitochondrial inner membrane whose import involves well-defined steps (Pfanner, N., and Neupert, W. (1987) J. Biol. Chem. 262, 7528-7536). Analysis of the in vitro import of gene fusion products containing ADP/ATP carrier (AAC) sequences at the amino terminus and mouse dihydrofolate reductase (DHFR) at the carboxyl terminus indicates that the first 72 amino acids of the soluble carrier protein, a hydrophilic region of the protein, are not by themselves sufficient for initial binding to the AAC receptor on the mitochondrial surface. However, an AAC-DHFR gene fusion containing the first 111 residues of the ADP/ATP carrier protein exhibited binding to mitochondria at low temperature (2 degrees C) and internalization at 25 degrees C to a mitochondrial space protected from proteinase K in the same manner as the wild-type ADP/ATP carrier protein. The AAC-DHFR protein, in contrast to the wild-type AAC protein imported into mitochondria under optimal conditions, remained extractable at alkaline pH and appeared to be blocked at an intermediate step in the AAC import pathway. Based on its extraction properties, this AAC-DHFR hybrid is proposed to be associated with a proteinaceous component of the import apparatus within mitochondria. These data indicate that the import determinants for the AAC protein are not located at its extreme amino terminus and that protein determinants distal to the first 111 residues of the carrier may be necessary to move the protein beyond the alkali-extractable step in the biogenesis of a functional AAC protein.

Fatty acid synthetase from Saccharomyces cerevisiae is a multifunctional enzyme which catalyzes the synthesis of long chain fatty acids from acetyl- and malonyl-CoA. The enzyme is composed of two nonidentical subunits, alpha (Mr = 212,000) and beta (Mr = 203,000), which are coded for by two unlinked genes FAS2 and FAS1, respectively. Individual yeast strains containing mutations in either of the FAS genes were transformed with a bank of yeast DNA sequences in the vector YEp13. Plasmids YEpFAS1 and YEpFAS2 were selected by their ability to complement the fas1 or fas2 mutations, respectively. Additionally, we utilized an immunologic screening of a second yeast DNA bank and selected two clones 33F1 and 102B5 which produce antigenically reactive material to anti-yeast fatty acid synthetase antibodies. Through Southern hybridization experiments and restriction endonuclease mapping, a region of 5.3 kilobase pairs of 33F1 was shown to be homologous with YEpFAS1, and a span of 3.4 kilobase pairs of 102B5 was homologous with YEpFAS2. These experiments identify the yeast DNA sequences cloned into 33F1 as originating from the FAS1 gene and those DNA sequences in 102B5, from the FAS2 gene.