Abstract. The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. Moreover, inhibition of prelamin A processing by mevinolin treatment is rapidly reversed by the addition of exogenous mevalonate. Processing of prelamin A is, therefore, dependent on isoprenoid metabolism. Analysis of the conversion of prelamin A to lamin A by two independent methods, immunoprecipitation and two-dimensional nonequilibrium pH gel electrophoresis, demonstrates that a precursor-product relationship exists between prelamin A and lamin A. Analysis of R,S-[5-3H(N)]mevalonate-labeled cells shows that the rate of turnover of the isoprenoid group from prelamin A is comparable to the rate of conversion of prelamin A to lamin A. These results~ suggest that during the proteolytic maturation of prelamin A, the isoprenylated moiety is lost. A significant difference between prelamin A processing, and that of p21 ra~ and the B-type lamins that undergo isoprenylationdependent proteolytic maturation, is that the mature form of lamin A is no longer isoprenylated.T HREE major polypeptides termed lamins A, B, and C (14, 17) comprise the nuclear lamina (1, 11,16,27,28) of most mammalian somatic cells. Individual lamins are substrates for posttranslational modifications that include cell cycle-dependent phosphorylation (7,15,37,42,52), methyl-esterification (9), and covalent modification with a mevalonate-derived product (4,6,34,47,49,50,58). The transient depolymerization of lamins, an event that is associated with the hyperphosphorylation of lamins (7,14,15,37,42) during mitosis, has been suggested to play a key regulatory role in the concomitant dissociation and reformation of the nuclear envelope. Membrane association appears to be an important function of the mevalonate-derived modifications to lamins in light of the apparent nuclear membrane localization of mammalian lamin B (7, 15, 18, 31), chicken lamin B2 (55), and the lamin A precursor polypeptide (22), all of which are modified by isoprenoids. The isoprenoid substituent of lamin B(lla), p21 ra~ (8, 46), and yeast polypeptide pheromones (3,23,25,39,45) has been identified as farnesyl. Gerace et al. (17) have shown that in CHO cells, prelamin A becomes NP-40 insoluble within 5 min of synthesis, and that it is in this lamina-associated environment that processing to mature lamin A occurs. We have previously reported (4) that this 74...

Abstract. We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [~4C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [~4C]mevalonate-labeled CHO cells is lamin B. The more basic 74-kD isoprenylated nuclear protein is similar in molecular mass and isoelectric pH variants to the lamin A precursor polypeptide reported by others. Starving cells for mevalonate results in a dramatic accumulation of a polypeptide that comigrates on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels with the 74-kD isoprenylated protein. The 70-kD isoprenylated protein, which is resolved on NEPHGE gels as being higher in molecular mass and slightly more basic than lamin B, has not yet been identified. T HE nuclear lamina, nuclear pore complex, and the inner and outer nuclear membranes are the major structural components of the nuclear envelope. Sequential extraction of isolated nuclear envelopes with nucleases, nonionic detergents, and high salt yields an insoluble fraction enriched in the structure known as the nuclear lamina (12,18,25). The three major polypeptide components of nuclear lamina from mammalian cells are designated lamins A, B, and C (16), and comprise •75% of the lamina mass (17). Lamins have been described in a range of vertebrates and invertebrates (15,19,22,38). Although there are variations in the type of lamins present in the nuclear lamina of some species, these proteins appear to be reasonably well conserved (22,38). Additional protein components of the lamina are also present in insect, avian, and rodent cells. These include lamin B subtypes or variants (10,30,31,48), precursor forms of lamins A and B (19, 26, 32), and Drosophila nuclear lamin isoforms (51).At mitosis, lamins undergo a transient and reversible depolymerization (6, 16, 52). During this process, lamins A and C become soluble and nonmembrane associated (6, 16). In contrast, lamin B remains associated with nuclear membrane vesicles (6,17,25,29), a phenomenon that may be due to hydrophobic properties of lamin B itself (19). Newly synthesized lamin A has been reported to exist as a short-lived precursor molecule (19,26,32,40). Lamin A appears to be This is contribution No. 916 of the Eleanor Roosevelt Institute for Cancer Research, Inc. processed to a mature form after being inserted into a Triton X-100-insoluble, membrane-associated fraction (19,32,40).We are currently investigating the subcellular distribution of a class of proteins that are ...

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The cost-effectiveness of identification and repair of vehicles with excessive carbon monoxide emissions was investigated over the 1991-92 winter period in Provo, Utah. This pilot program used on-road remote sensing at two locations to identify repeat gross polluting vehicles. The owners of gross polluters observed at one of the locations were solicited for a free repair program carried out under the direction of the Utah Valley Community College, Auto-Diesel Division. The same two locations were revisited after the repair program had terminated and the vehicle emissions were remeasured. More than 17,000 measurements of over 10,000 individual vehicles were obtained. As observed elsewhere, half of the total carbon monoxide was emitted by only about ten percent of the vehicles. Solicitation letters were sent to 114 owners and 47 vehicles were recruited and repairs attempted. Df the 47 vehicles, 28 were remeasured when the site was revisited at the end of the program. They had improved their measured on-road emissions by more than 50 percent. The vehicles which were identified as gross polluters at the second location-but were not notified of their status-were used as a control group. Their emissions were also reduced, as expected, but only by 14 percent. This pilot program demonstrates carbon monoxide emissions reduction at a cost effectiveness of $200 per ton, not including the cost benefits of gas mileage improvement. Two hundred dollars per ton is lowerthan many current or proposed mandated programs. The owner's repair cost would more than pay for itself in terms of improved fuel economy. The program would also generate the on-road fleet emissions data necessary to evaluate its effectiveness. Without such data, a program is forced to rely upon computer modeling with its known limitations. Most vehicles show CO/CO 2 and HC/ CO 2 ratios close to zero. When CO/CO 2 ratios greater than zero are observed, the engine must be operating with a fuel rich air/fuel ratio. In the case of a large HC/ CO 2 ratio, a fuel lean air/fuel ratio which is

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