Replicate-based noise corrected correlation for accurate measurements of colocalization.

It is widely recognized that the accuracy of colocalization measurements is dependent upon the quality of the source images. We demonstrate that, as the image quality increases, the measured colocalization, using the Pearson and Spearman rank correlation coefficients, approaches the true colocalization asymptotically. This means that in practice it is difficult to obtain images of sufficient quality for accurate measurements. We introduce the replicate-based noise corrected correlation (RBNCC) which aligns the measured colocalization with the true colocalization: a noise measurement is made for each fluorophore from a pair of replicate images, the two noise measurements are combined to generate a correction factor which is applied to the measured colocalization between the two fluorophores. In consequence, accurate measurements can be obtained even with noisy images, making RBNCC especially attractive for live imaging. Even with images of apparently good quality we found an average discrepancy of about 20% between the measured and corrected colocalization. A case is made for using the Spearman rank coefficient instead of the Pearson coefficient to measure colocalization.

Protein prenylation in spinach chloroplasts.

Protein prenylation in plants was studied by in vivo metabolic (3)H-mevalonate labeling in combination with a range of protein synthesis inhibitors. In spinach cotyledons, this posttranslational protein modification was found to be divided into two categories, one representing the conventional prenylation involving farnesyl and geranylgeranyl groups bound to cysteine residues via thioether linkages. This category revealed a similar pattern of prenylated proteins to that observed in mammalian cells and depends on nuclear gene expression. The other category was shown to represent a type of prenylation confined to chloroplasts. It depends on plastid gene expression and does not involve a thioether bond. The modifying isoprenoid could be released from the chloroplastic polypeptides by alkaline treatment and was identified as phytol upon GC-MS analysis. The phytol could readily be derived from all-trans-[(3)H]farnesol, which, like all-trans-[(3)H]geranylgeraniol, was taken up by the cotyledons, resulting in incorporation of radiolabel into proteins.

Synthesis of mevalonate pathway lipids in fibroblasts from Zellweger and X-linked ALD patients.

Fibroblasts were cultured to determine the involvement of peroxisomes in cholesterol and dolichol synthesis. For this purpose, the behavior of cells from patients with Zellweger syndrome, with X-linked adrenoleukodystrophy, and from nondiseased control subjects was studied. Cells both after pretreatment with mevinolin and without pretreatment were incubated in a medium containing [3H]-mevalonate. In fibroblasts from patients with peroxisomal defects, the cholesterol content and mevalonate incorporation into cholesterol were decreased by 10-20% in comparison with control cells. Mevinolin pretreatment decreased the incorporation rate of [3H]-mevalonate into cholesterol but increased the labeling of ubiquinone and dolichol both in diseased and control cells. Squalene synthase activity was unchanged, whereas the activity of farnesyl-pyrophosphate synthase was increased in the diseased states. The results show that in patients with peroxisomal deficiency neither the amount nor the rate of synthesis of cholesterol and dolichol is reduced to any greater extent.

Subcellular distribution of farnesyl protein transferase in rat liver.

Farnesyl protein transferase (FPT) activity was measured in rat liver subcellular fractions by using an unspecific acceptor for the farnesyl groups. The highest specific activity was found in mitochondria and it exceeded that of the microsomes three-fold. Considerably lower specific activities were found in the nuclei and cytosol. Further subfractionation revealed that the mitochondrial FPT activity is located in the matrix. The beta-subunit of the mitochondrial enzyme has an apparent molecular mass of 46 kDa, which is similar to its cytosolic counterpart. The results suggest that protein farnesylation can take place in a number of subcellular organelles.

In vivo prenylation of rat proteins: modification of proteins with penta- and hexaprenyl groups.

In vivo protein prenylation was studied in newborn rats by repeated injections of [3H]mevalonate. The highest level of protein-bound mevalonate metabolites was found in the kidney, but incorporation was observed in all organs studied. After fluorography of SDS-polyacrylamide gel electrophoresis-separated polypeptides, labeling was found in the 21- to 28-kDa molecular mass region and, after prolonged exposure of the film, additional bands at both higher and lower molecular masses could be detected. Protein prenylation in the kidney increased during the first 5 days after birth, whereas that in the liver reached a maximum on the fourth day. After methyliodide treatment of the prenylated proteins, farnesol, geranylgeraniol, and two larger isoprenoids, pentaprenol and hexaprenol, were released. In the liver, the ratio of protein-bound geranylgeraniol to farnesol increased from 2 to 4.5 during the first 5 days after birth. Upon subfractionation of the kidney, the highest level of labeling was found in mitochondria and microsomes. When the mitochondria were subfractionated into outer membranes, intermembrane space and an inner membrane/matrix fraction, the labeling pattern of prenylated polypeptides differed in all fractions. The results demonstrate that in vivo labeling of rats can be performed to study the extent, type, and distribution of protein prenylation.

Chloroplastic prenylated proteins.

By in vivo [3H]mevalonate labelling of spinach combined with biochemical analysis, evidence is provided for the existence of protein prenylation in chloroplasts. Approximately 20 prenylated polypeptides were resolved by SDS-PAGE followed by autoradiography. Thermolysin treatment of intact chloroplasts revealed that about 40% of the prenylated polypeptides were associated with the cytoplasmic surface of the outer envelope membrane. The remaining portion was present in thylakoids and/or the inner envelope membrane. The majority of the prenylated polypeptides were associated with larger membrane protein complexes. A farnesyl protein transferase activity was found to be associated with the thylakoid membrane.

Protein prenylation in spinach--tissue specificity and greening-induced changes.

Etiolated spinach seedlings, as well as petioles and blades of leaves of green seedlings, were labeled with [3H]mevalonate to study protein prenylation in several plant developmental stages. The polypeptide prenylation pattern of the leaf petiole and the leaf blade differed considerably, although some prenylated proteins were present in both tissues. During greening several prenylated polypeptides in the 30- to 46-kDa molecular mass region and two at 15 kDa became more abundant, while others in the 21.5- to 30-kDa region and one at 62 kDa showed a relative decrease. However, the relative amount of several of the prenylated polypeptides did not appear to be altered during the greening process. In etiolated seedlings, more thioether-linked farnesol than geranylgeraniol was found, whereas in seedlings grown under normal light conditions the converse situation prevailed.

Identification of spinach farnesyl protein transferase. Dithiothreitol as an acceptor in vitro.

Spinach seedlings were found to contain farnesyl protein transferase. The enzyme is activated by Zn2+, but not by Mg2+. The pH optimum is approximately 7.0 and maximal activity is obtained at 40-45 degrees C. The apparent Km for the farnesyl diphosphate substrate is 7 microM. Western blotting of soluble proteins with an antiserum raised against mammalian farnesyl protein transferase demonstrated a specific cross-reactivity with the spinach enzyme. The antiserum preferentially recognises the beta-subunit of the heterodimeric farnesyl protein transferase, and the corresponding spinach polypeptide has a molecular mass of 42 kDa on SDS/PAGE. The enzyme can employ dithiothreitol as an acceptor for the farnesyl moiety and catalyses the formation of a thioether linkage between these substrates. On the basis of this discovery, a new method was developed utilising the hydrophobicity of the reaction product, and its interaction with poly(propylene). During in vivo labelling, the plants took up dithiothreitol, which inhibited the incorporation of [3H]mevalonate metabolites into proteins, indicating that dithiothreitol might be isoprenylated in vivo as well as in vitro. However, isoprenylation of some proteins remains unaffected by dithiothreitol suggesting the existence of different isoprenylation mechanisms. Thus, it is demonstrated that plants possess farnesyl protein transferase, which resembles its mammalian and yeast homologues.

Distribution of branch point prenyltransferases in regions of bovine brain.

Bovine brains contain large amounts of isoprenoid compounds and the enzymes involved in their biosynthesis were investigated. Ten different regions were dissected from fresh bovine brains and, in addition, fractions from cerebellum, spinal cord, and hypophysis were obtained. The cholesterol concentration was found to be approximately 8 mg/g in the cortex regions and three times higher in the pons, medulla oblongata, and white matter. Dolichol concentration varied between 8 and 40 micrograms/g in the different tissues, and ubiquinone was found at a lower level, which varied between 3 and 25 micrograms/g. Farnesylpyrophosphate synthase activity in cytosolic fractions from various regions exhibited only a twofold variation, whereas geranylgeranyl pyrophosphate synthase displayed larger differences, being particularly rich in the pons, medulla oblongata, white matter, and spinal cord. Squalene synthase activity was lowest in the thalamus and threefold higher in the pons. Determination of specific activity based on cholesterol content revealed that enzyme activities in various regions are not related to the actual lipid amount present. Both cis- and trans-prenyltransferases exhibited similarities in their regional distribution showing up to 20-fold differences in activity. Thus, it appears that the mevalonate pathway lipids and the various branch point enzymes involved in their syntheses vary greatly in different brain regions and are subjected to separate regulation.

Isoprenylation of plant proteins in vivo. Isoprenylated proteins are abundant in the mitochondria and nuclei of spinach.

Protein isoprenylation in vivo is demonstrated using spinach seedlings labeled with [3H]mevalonate. This report provides evidence for the occurrence of a large number of isoprenylated proteins in plants. Seedlings, without roots, were labeled quantitatively through the cut stem. Mevinolin treatment of the seedlings resulted in increased incorporation of radiolabel into proteins. Approximately 30 labeled bands could be detected after autoradiography of SDS-polyacrylamide gel electrophoresis-separated polypeptides, ranging in molecular mass from 6 to 200 kDa. Methyl iodide hydrolysis resulted in the release of covalently bound farnesol, geranylgeraniol, phytol, and some unidentified isoprenoid compounds from mevalonate-labeled proteins. It was found that all cellular fractions contained some isoprenylated proteins, although most were located in the mitochondria and nuclei. Subfractionation of the nucleus revealed that the majority of isoprenylated proteins in this compartment were components of the nuclear matrix. The results demonstrate that in vivo labeling of a complex organism can be performed using a plant system in order to study protein isoprenylation and distribution of modified proteins in different cellular compartments.

Regulation of coenzyme Q biosynthesis.

The side-chain moiety of coenzyme Q is synthesized by a trans-prenyltransferase present in microsomes. Condensation of this moiety with the precursor ring takes place in the Golgi system. The enzymes involved, as well as the cytosolic geranylgeranyl-PP synthase, are regulated in an independent fashion. When the size of the farnesyl-PP pool is decreased or increased by employing appropriate inhibitors, the rate of CoQ synthesis is modified accordingly, indicating the dependence of trans-prenyltransferase activity on the level of intracellular substrate concentrations. Administration of peroxisome proliferators elevates CoQ concentrations not only in blood, but also in various tissues. Thus, it may be possible in the future to selectively increase CoQ concentrations in certain organs, without increasing the level of cholesterol.