(Received for publication, September 20, 1995; and in revised form, October 26, 1995)
From the
We have recently demonstrated that mevalonate kinase and
farnesyl diphosphate (FPP) synthase are localized predominantly in
peroxisomes. This observation raises the question regarding the
subcellular localization of the enzymes that catalyze the individual
steps in the pathway between mevalonate kinase and FPP synthase
(phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and
isopentenyl diphosphate isomerase). These enzyme are found in the
100,000 g supernatant fraction of cells or tissues and
have been considered to be cytoplasmic proteins. In the current
studies, we show that the activities of mevalonate kinase,
phosphomevalonate kinase, and mevalonate diphosphate decarboxylase are
equal in extracts prepared from intact cells and selectively
permeabilized cells, which lack cytosolic enzymes. We also demonstrate
structure-linked latency of phosphomevalonate kinase and mevalonate
diphosphate decarboxylase that is consistent with a peroxisomal
localization of these enzymes. Finally, we show that cholesterol
biosynthesis from mevalonate can occur in selectively permeabilized
cells lacking cytosolic components. These results suggest that the
peroxisome is the major site of the synthesis of FPP from mevalonate,
since all of the cholestrogenic enzymes involved in this conversion are
localized in the peroxisome.
Recently, it has been demonstrated by our group and others that
peroxisomes contain a number of enzymes involved in cholesterol
biosynthesis that previously were considered to be cytosolic or located
exclusively in the endoplasmic reticulum. Peroxisomes have been shown
to contain acetoacetyl-CoA thiolase(1, 2) ,
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) ()synthase(3) , HMG-CoA
reductase(4, 5, 6) , mevalonate
kinase(7, 8) , and most recently farnesyl diphosphate
(FPP) synthase(9) . Both mevalonate kinase and FPP synthase
seem to be localized predominantly, if not exclusively, to peroxisomes (8, 9) .
The demonstration that mevalonate kinase and FPP synthase are localized predominantly in peroxisomes (8, 9) raises the question regarding the localization of the enzymes that catalyze the individual steps in the pathway between mevalonate kinase and FPP synthase (phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase). Based on results obtained from fractionation studies, these enzymes are believed to be localized in the cytosol. However, recent data have shown that the activities of these enzymes as well as mevalonate kinase and FPP synthase are significantly reduced in liver tissue obtained from patients with peroxisome-deficient diseases (Zellweger syndrome and neonatal adrenoleukodystrophy), thus indicating a peroxisomal localization(9) .
We have routinely employed three different methods to study subcellular localization of proteins: (i) analytical subcellular fractionation and measurements of enzyme activities, (ii) immunoblotting of the protein in the isolated fractions with a monospecific antibody, and (iii) immunoelectron and immunofluorescence microscopy. In the studies demonstrating the peroxisomal localization of mevalonate kinase and FPP synthase(8, 9) , it was shown that analytical subcellular fractionation of liver and measurements of enzyme activities are not sufficient to determine intracellular localization due to the release of these enzymes in the cytososlic fraction from peroxisomes during the isolation of the organelle. Immunoelectron and immunofluorescence microscopy studies with specific antibodies to these enzymes were critical in determining the correct subcellular localization. To our knowledge antibodies to phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase are not currently available. Hence, in order to study the subcellular localization of these enzymes, we have selected to use permeabilized cells, which retain their organelle integrity yet lack cytosolic components. Permeabilized cells have been used successfully in a number of different studies dealing with subcellular function and localization.
To test whether only the plasma membrane was disrupted and the cell organelles remained intact, we determined various marker enzyme activities in both permeabilized and intact cells. Table 1illustrates that there was no significant difference between the two groups in the total activity per plate of esterase (marker enzyme for endoplasmic reticulum) and catalase (marker enzyme for peroxisomes), whereas phosphoglucose isomerase (a marker enzyme for cytosolic fraction) was measurable in the control cells and was not detectable in the permeabilized cells. Furthermore, the protein concentration in permeabilized cells was approximately 30-50% less than that of intact cells. This corresponds to the protein content of cell cytosol. However, to further demonstrate that the peroxisomal, ER and mitochondrial compartments of cells remain intact after permeabilization while the cytosolic contents disappear, we employed an additional method. Control and permeabilized cells grown on coverslips were treated in parallel with antibodies to various marker enzymes as described under ``Experimental Procedures.'' Fig. 1illustrates the immunofluoresence pattern obtained for cytosol, peroxisomes, ER, and mitochondria in control cells and digitonin permeabilized cells. Panel A in Fig. 1shows cytosolic labeling in intact CV-1 cells, whereas the permeabilized CV-1 cells in panel B are devoid of cytosolic labeling. The bright fluorescence in the center of the cell is due to autofluorescence of the nucleus. In panels C and D, the cells were labeled for peroxisomal proteins using an antibody made against the peroxisomal targeting signal (SKL at the C terminus)(20) . A uniform punctate pattern characteristic of peroxisomal labeling is observed in both the intact cells (panel C) as well as in the permeabilized cells (panel D). Additionally, the pattern of ER labeling is similar in control cells (panel E) and permeabilized cells (panel F). Cells in panels G (control) and H (permeabilized) demonstrate that the mitochondrial membrane also remains intact during selective permeabilization with digitonin. Taken together, these results demonstrate that the plasma membrane of CV-1 cells can be selectively permeabilized with low concentrations of digitonin, resulting in the release of cytosolic proteins while maintaining organelle integrity.
Figure 1: Selective permeabilization of the plasma membrane releases cytosolic components but maintains the integrity of subcellular organelles. Control and permeabilized cells grown on coverslips were treated in parallel with antibodies to various marker enzymes as described under ``Experimental Procedures.'' Panels A, C, E, and G illustrate control cell labeling; panels B, D, F, and H represent labeling of permeabilized cells. Panel A shows cytosolic labeling in intact cells; panel B illustrates the absence of cytosolic labeling in permeabilized cells. The bright fluorescence in the center of the cell is due to autofluorescence of the nucleus. In panels C and D, the cells were labeled for peroxisomal proteins, in panels E and F for ER proteins, and in panels G and H for mitochondrial proteins.
Figure 2: The release of catalase activity from peroxisomes as a function of time after selective permeabilization of the plasma membrane. Cells were permeabilized and then assayed for catalase activity in the presence and absence of 0.1% Triton X-100. The graph represents mean ± S.E. values of three sets of experiments. Values are expressed as percent of total catalase activity at each time point as measured in the presence of Triton X-100.
Results of these experiments are shown in Table 3. The mean value for control cells was 4287 dpm/plate and for permeabilized cells 3026 dpm/plate. These two means are not significantly different. No conversion of mevalonate to cholesterol was observed if ATP and/or NADPH were omitted from the reaction buffer (data not shown). These results indicate that the cytosolic fraction of cells is not necessary for the biosynthesis of cholesterol from mevalonate.
In summary, the current report demonstrates that mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase activities in extracts prepared from intact cells are equal to those of selectively permeabilized cells that lack cytosolic enzymes. We also demonstrate structure-linked latency of phosphomevalonate kinase and mevalonate diphosphate decarboxylase that is consistent with a peroxisomal localization of these enzymes. These results in combination with the previous observation that mevalonate kinase and FPP synthase are predominantly localized to peroxisomes(8, 9) , suggest that all of the cholesterogenic enzymes involved in the conversion of mevalonate to FPP are localized to the peroxisome. This conclusion is further supported by the direct finding that cholesterol biosynthesis from mevalonate can occur in selectively permeabilized cells lacking cytosolic components and indirectly by the previous observation that all the required enzymes for the conversion of mevalonate to FPP are significantly reduced in tissue obtained from patients with Zellweger syndrome and neonatal adrenoleukodystrophy(9) .
The isoprenoid biosynthetic pathway is unrivaled in nature for the chemical diversity of the compounds it produces. FPP is a key intermediate that serves as a substrate for a number of critical branch-point enzymes including the synthesis of squalene, cholesterol, farnesylated and geranylgeranylated proteins, dolichols, coenzyme Q, and the isoprenoid moiety of heme a. Thus, the regulation and levels of FPP are important since large perturbations in FPP could alter the flux of isoprenoid compounds down the various branches of the pathway.
If indeed, the majority of the cell's FPP is produced in the peroxisomes, this means that FPP and/or farnesol has to be transported out of peroxisomes for further metabolism. Since phosphorylated products of mevalonate are not able to cross the peroxisomal membrane, it is likely that FPP may also be impermeable. Therefore, is FPP first converted to farnesol in the peroxisome, and then is freely diffusible out of the organelle? Or is there a transport/binding protein that facilitates the movement of these intermediates? What determines where FPP is utilized? What regulates FPP conversion to farnesol and farnesol conversion to dicarboxylic acids? These are important questions that need to be addressed in order to understand the regulation of FPP/and or farnesol.
It is significant to note two recent studies demonstrating the potential importance of farnesol in regulation of cellular function (21, 22) . In the first study, farnesol has been identified as the non-sterol derivative that can initiate and promote the degradation of HMG-CoA reductase in permeabilized cells(21) . In the second study, an orphan nuclear receptor named farnesoid X-activated receptor (FXR) is described that is activated by farnesol(22) . Thus, FXR provides an example of a vertebrate transcription factor that is regulated by an intracellular metabolite (farnesol) and may indicate the existence of a novel vertebrate-signaling pathway(22) . The FXR target genes remain to be identified.