Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart.

Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called 'CAAX box' at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.

Yeast CAL1 is a structural and functional homologue to the DPR1 (RAM) gene involved in ras processing.

A 2.3-kilobase pair DNA fragment of the yeast CAL1 gene was cloned by complementation of the cal1-1 mutation, which causes a defect in nuclear division and bud formation (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1984) Mol. & Gen. Genet. 193, 389-394). Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) encoding a polypeptide of 376 amino acids. Comparative analysis of the predicted amino acid sequence has shown that the CAL1 product has similarity to two yeast proteins: the DPR1 (RAM) gene product that is involved in processing of ras protein at the farnesylation step, and the essential ORF2 protein whose structural gene has a head-to-head arrangement with PRP4, which is involved in mRNA processing. Functional homology between CAL1 and DPR1 has also been suggested from genetic evidence that multiple copies of the CAL1 gene suppress the growth defects of a dpr1 null mutant at high temperature. This suppression is Ca(2+)-dependent, since it was not observed in complete medium containing 200 microM CaCl2 but was apparent in medium containing 100 mM CaCl2. From sequence analysis of the cal1-1 mutation, together with the alignment of the three gene products, we have concluded that the conserved Gly328 in the C terminus is important for activity. We suggest that the CAL1 protein participates in a ras-like C-terminal modification of proteins involved in nuclear division and bud growth.

Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins.

Ras proteins are post-translationally modified by farnesylation. In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein. RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not. A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity. In addition, another mutant, ram2, also was defective in the transferase activity. These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast.

Structure and expression of yeast DPR1, a gene essential for the processing and intracellular localization of ras proteins.

The ras proteins represent a unique example of membrane proteins which apparently do not utilize the secretory pathway for their membrane localization. Instead, it is believed that palmitic acid, covalently attached to the protein, acts as an anchor to the membranes. Recent identification of yeast mutants defective in the processing of the ras proteins has provided a novel approach for defining these biosynthetic processes. We report here the characterization of yeast DPR1, a gene essential for the processing of the ras proteins. The sequence of the gene indicates that it encodes a protein of 431 amino acids which contains no significant homology with any known proteins. It is a relatively hydrophilic protein with no apparent hydrophobic stretches. The C-terminal half of the encoded protein has an unusually high content of cysteine. The DPR1 gene product has been identified in a cell-free translation system as a protein having an apparent molecular weight of 43 kd. This represents the first step in the investigation of a novel protein-processing pathway, one that is distinct from the secretory pathway.

Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification.

Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttraslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by the dprl mutation which has recently been shown to affect the processing of yeast RAS proteins. To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.

Expression of the Bacillus subtilis polC gene in Escherichia coli.

We have cloned a 14 kb DNA segment containing the coding sequence (polC) for DNA polymerase III (PolIII) from the Bacillus subtilis chromosome. The plasmid carrying the sequence, pRO10, directs the synthesis of the 160 kDa PolIII protein and three additional polypeptides in Escherichia coli maxicells from strain CSR603. A set of deletion derivatives of pRO10 was constructed in vitro. The location of the PolIII coding sequence and the direction of transcription through the polC gene were determined by analysis of the truncated polypeptides observed in extracts of CSR603 transformants. Two HindIII segments subcloned from pRO10 were found to contain promoter sequences which function in E. coli and in vegetative phase B. subtilis cells. The location of the promoter sequence was determined with respect to the polC gene. The B. subtilis polC gene did not complement the temperature-sensitive defect of an E. coli PolIII mutant (dnaE486). The presence of the complete B. subtilis polC gene on a multicopy plasmid inhibited the growth of E. coli cells.

Medicalization and its discontents.

This paper raises the question of the ethically proper balance in health care policy between the medical-clinical-high technology model of health service and the grass-roots, community based or traditional models of care. Paradoxical imbalances between the two approaches are traced to political, economic or prestige factors. Case studies examined include the hospitalization of non-contagious leprosy patients while protecting the anonymity of AIDS-infected prostitutes, medical resistance to the adoption of a clinical role by Community Cancer Centers, and the continued preference in some quarters for elaborate (and often delayed) hospital treatment for such problems as infant diarrhea, despite the availability of much simpler solutions, as in the case of the widely successful oral rehydration therapy. A balanced approach to world health problems, we argue, rests not on inflationary lowering of health care standards to achieve nominal victories, nor on stainless steel high technology panaceas but on mobilizing resources around human needs.

Prevention--how misuse of a concept undercuts its worth.

Some health leaders and researchers have launched mass prevention programs without sound biomedical groundwork. They have oversold the benefits of prevention and underestimated the secondary effects. Some have forced nonmedical concerns into the medical model. Others have blurred the distinctions between prevention and other measures such as screening or therapy. Some have transferred responsibility for disease to the victim. A few have imputed magical powers to certain symbols of prevention, in order to create an illusion of control.

KIE: Examples are cited to argue that some mass prevention programs have been oversold. The National Cancer Institute and American Cancer Society's promotion of dietary changes to reduce cancer risk has spawned risky diets promoted by food faddists. Overuse of hysterectomy and estrogen replacement therapy and an overzealous swine flu immunization campaign have caused needless mortality and morbidity from complications. Promoting adolescent contraception as a means to reduce child abuse represents an overextension of the idea of preventive health measures into the nonmedical sphere, as do physicians' campaigns that amplify children's fears of nuclear disaster. Public confusion about the distinction between therapy and prevention has been fostered by groups soliciting funds for cancer research, while some health promotion campaigns have contributed to a blame-the-victim mentality and to the imputing of magical powers to certain prevention symbols.

The ovrselling of genetic anxiety.

KIE: Mass screening programs to detect carriers of Tay Sachs disease in the American Jewish population are described and criticized from an ethical standpoint. Educational programs promoting screening often trigger long-lasting anxiety among members of the target population. Other ethical problem areas include the co-opting of community resources and the dangers of stigmatization of carriers. In addition, cost-benefit analyses have been narrowly construed and have failed to demonstrate any clearcut advantage to mass screening. Finally, alternative approaches have been proposed to achieve the goal of preventing Tay Sachs births through individual counseling of high-risk couples.^ieng