(Received for publication, July 9, 1996, and in revised form, September 17, 1996)
From the Department of Biology, Faculty of Science,
Ochanomizu University, Tokyo 112, Japan and ¶ Department of
Biophysics and Biochemistry, Faculty of Science, University of Tokyo,
Tokyo 113, Japan
To know the very early events occurring after heat shock, the changes of membrane lipids were examined. Heat stress induced the production of a certain glycolipid in the myxoamoebae of Physarum polycephalum in a few minutes. The purified glycolipid was determined to be a poriferasterol monoglucoside by structural studies that was previously reported to be expressed during the differentiation of Physarum cells from haploid myxoamoebae to diploid plasmodia (Murakami-Murofushi, K., Nakamura, K., Ohta, J., Suzuki, M., Suzuki, A., Murofushi, H., and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723). The activity of UDP-glucose:poriferasterol glucosyltransferase (Murakami-Murofushi, K., and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415) was also expressed rapidly after heat shock. Thus, the activation of sterol glucosyltransferase and the production of sterol-glucoside were considered to be important events that were involved in the signal transduction system to induce some succeeding heat-shock responses, such as the synthesis of heat-shock proteins.
In response to environmental stresses, living organisms acquired the capability of recognizing such stresses and adapting themselves to various types of stress during evolution. They induce some proteins, so-called stress proteins or heat-shock proteins, to protect themselves from conditions unfavorable for their survival.
Under stress conditions such as heat shock, starvation, high salt, and high osmotic pressure, haploid myxoamoebae of a true slime mold, Physarum polycephalum, retracted their pseudopodia and changed shape into a disk-like form, then they constructed cell walls to form their dormant form, microcysts. These morphological changes were associated with changes in the intracellular distribution of actin filaments. Synthesis of a novel stress protein, p66, was induced within 15 min after heat shock. Because this protein coprecipitated with polymerized actin in vitro and colocalized with short bundles of actin filaments in vivo, it may have participated in the change of actin distribution associated with heat-inducible microcyst formation. However, p66 was not induced when diploid plasmodia of Physarum were exposed to heat shock, so this protein is considered to be specifically expressed during microcyst formation in the haploid stage of Physarum (3). The structure of the p66 gene, the biological functions of p66, and the regulation of actin-p66 binding are now being investigated in our laboratory.
In many organisms, the induction and properties of heat-shock proteins have been investigated in detail (4, 5), and many investigators are now working on this subject. However, very early events after heat shock have not yet been clarified. Because the plasma membrane may receive a heat shock first and then the signal may be transduced into the cell, we studied the change of membrane lipids after heat shock and demonstrated a rapid induction of a certain glycolipid and its synthesizing enzyme in myxoamoebae of P. polycephalum.
Myxoamoebae of a true slime mold, P. polycephalum, were grown on a lawn of bacteria, Aerobacter aerogenes, in a nutrient agar medium in the dark at 24 °C (6). For heat shock, they were incubated at 40 °C.
ChemicalsSephadex A-25 was obtained from Pharmacia Fine
Chemicals. Silica gel 60 thin layer chromatography
(TLC)1 plates and high-performance TLC
plates of silica gel 60 were from Merck, and 3% OV-101 on Shimalite W
(80-100 mesh) was from Shimadzu (Kyoto, Japan). J & W megabore column
DB-1 (15 m × 0.53 mm; film thickness, 1.5 µm) and J & W
capillary column DB-1 (15 m × 0.26 mm; film thickness, 0.25 µm)
were from J & W Scientific. Egg phosphatidylcholine, cholesterol,
ergosterol, stigmasterol, campesterol, sitosterol, and - and
-glucosidase were purchased from Sigma, and
poriferasterol was isolated from plasmodia of P. polycephalum as described previously (7, 8). Poriferasterol monoglucoside, which is present in the plasma membrane of
Physarum plasmodia, was purified as reported previously
(8).
Extraction and purification of an expressed glycolipid was performed essentially according to the procedure described previously by a combination of gel filtration by Sephadex A-25 and preparative TLC using silica gel 60 plates in solvent system I and II (I, chloroform:methanol:water (60:40:9, v/v); II, chloroform:methanol:acetone:acetic acid:water (10:2:4:2:1, v/v)) (1).
TLCLipids were analyzed by TLC in solvent system I and II, and glycolipids were visualized by spraying with 2% orcinol in 2 N H2SO4 followed by heating at 125 °C. Other color-developing reagents, such as resorcinol-HCl for sialic acid, azure A-H2SO4 for sulfolipids, ninhydrin for amino acids, 0.05% ferric chloride in 5% each of acetic acid and sulfuric acid for sterols, and molybdenum blue reagent for phosphate-containing compounds, were also tested (1). Solvent systems I and II were also used for two-dimensional TLC.
Gas-Liquid Chromatography (GLC)For the detection of components of a purified glycolipid, homogeneous substance was methanolized, and the resultant methyl glycoside and sterol were trimethylsilylated and analyzed by GLC on a column of 3% OV-101 as described (1).
Fast Atom Bombardment Mass SpectrometryFast atom bombardment mass spectrometry for an intact glycolipid was carried out on a JEOL DX-303 mass spectrometer under the described conditions (1).
Enzymatic HydrolysisThe glycolipid was dissolved in 50 mM sodium citrate, pH 5.0, and treated with - or
-glucosidase as described (1).
The assay of this transferase was performed essentially by the procedure of Wojciechowski et al. (9, 10) with slight modification (2).
Other MethodsColorimetric determination of hexose and sterol was performed according to Radin et al. (11) and Momose et al. (12), respectively.
When myxoamoebae were incubated at 40 °C, the
induction of a certain glycolipid was prominent soon after the
temperature shift. Physarum myxoamoebae were heat-treated at
40 °C for various periods, and crude lipid fractions that were
extracted from heat-shocked cells were analyzed by TLC. A certain
glycolipid, designated GL-X (Fig. 1), appeared just
after the temperature shift and increased in amount for about 10 min. A
content of this substance was maintained constantly for at least 60 min. The expression of this substance was followed by the induction of
stress protein p66 and some other heat-shock proteins and microcyst
formation as shown in Fig. 2.
A slight change of glycolipid C (Fig. 1) was also observed, but it was already present in significant quantities before the temperature shift. The chemical nature of glycolipids A-D and the meaning of the slight change of glycolipid C have not yet been studied.
Purification and Structural Studies of a GL-XGL-X was
purified as described under "Experimental Procedures," and the
purity of this substance was analyzed by TLC in different solvent
systems (I and II) and with some different color-developing reagents
for the detection of some biochemical compounds. GL-X showed a single
spot in each solvent system used and visualized with
orcinol-H2SO4 and ferric chloride solution. No
other reagents tested reacted with GL-X. Fig. 3 shows
the two-dimensional TLC analysis of GL-X visualized with orcinol
reagent (A) and ferric chloride reagent (B).
Hence, GL-X is shown to be composed of hexose and sterol.
The purified GL-X was subjected to a negative fast atom bombardment
mass spectrometry. The [M-1] ion at m/e 573 was obtained, then the molecular weight of GL-X was determined to be
574 (Fig. 4). This molecular weight is identical with
that of poriferasterol monoglucoside reported previously in
Physarum plasmodia (1).
GL-X was methanolized, and trimethylsilyl derivatives of methyl
glycoside and sterol were analyzed by GLC as shown in Figs. 5 and 6. From these results, the sugar
moiety and sterol moiety of GL-X were determined as glucose and
poriferasterol, respectively. No other components were detected by GLC
analysis under some different conditions with some different
columns.
Because GL-X was hydrolyzed with -glucosidase but not
-glucosidase (data not shown), the linkage of glucose
-poriferasterol was suggested. From a colorimetric determination of
glucose and sterol, a molar ratio of 1.0:1.1 was obtained.
Table I shows the RF values of GL-X and the standard poriferasterol monoglucoside that was isolated from plasmodia of P. polycephalum, and these values also supported the identification of GL-X as poriferasterol monoglucoside (Fig. 7).
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Induction of UDP-glucose:Poriferasterol Glucosyltransferase Activity in Heat-shocked Myxoamoebae
Endogenous
UDP-glucose:poriferasterol glucosyltransferase activities were assayed
in the homogenates of haploid myxoamoebae before and after temperature
shift from 24 °C to 40 °C. The enzyme activity was not detected
in the homogenate before heat shock, but an apparent activation of
UDP-glucose:poriferasterol glucosyltransferase was observed after heat
shock as shown in Fig. 8.
In this report, we showed a rapid expression of poriferasterol monoglucoside by heat shock at 40 °C in the haploid myxoamoebae of P. polycephalum. The enzyme UDP-glucose:poriferasterol glucosyltransferase was also activated rapidly after heat shock to form the above-mentioned glycolipid. From these findings, rapid production of steryl glucoside might be involved in the very early process of stress response of Physarum myxoamoebae.
Previously, we reported the expression of this substance during the differentiation of Physarum cells from haploid myxoamoebae to diploid plasmodia (1), and an expression of UDP-glucose:poriferasterol glucosyltransferase activity associated with the differentiation was also demonstrated (2). It takes about 1 week after mating for myxoamoebae to differentiate into plasmodia, but the steryl glucoside and its synthesizing enzyme appeared at an early stage of differentiation (1 day after the mating of haploid cells).
The steryl glucoside and its 6-O-acyl derivatives are known
as common constituents of higher plants (13, 14), and their functions
are considered to be metabolically active components of plant membrane
structure (15), intercellular transporters of sterols (16), or glucose
carriers through cell membranes (17, 18). In Physarum,
plasmodia are capable of growth in liquid or on agar media, but
myxoamoebae, except for the rare mutant strain Colonia (19),
can be cultured only on bacterial lawns. Myxoamoebae may not be able to
utilize glucose and other small molecules, but plasmodia are capable of
utilizing them as nutrients. Poriferasterol monoglucoside may have some
active functions in membranes showing such interesting properties.
A matingless mutant, Colonia strain, differentiates from myxoamoebae into plasmodia without any changes of nuclear DNA. When the cultivating temperature is reduced from 30 °C to 25 °C, the myxoamoebae differentiate into plasmodia without conjugation, and then haploid, not diploid, plasmodia are formed. We showed that the cells of this mutant strain contained poriferasterol monoglucoside and its synthesizing enzyme in both the myxoamoeboid and plasmodial stages. We also demonstrated that Colonia cells showed a slower rate of growth than that of wild-type ones on a lawn of bacteria, but they could survive and increase their cell number even in culture medium (20). This indicates that the Colonia myxoamoebae can uptake some small substances from the nutrient media as their energy source. Then the uptake of glucose and amino acids into myxoamoebae was measured. The results clearly showed a much higher uptake of D-glucose into Colonia cells than into wild-type cells. The uptake ability of amino acids into wild-type and mutant myxoamoebae was examined, and no difference between them was observed. Because almost no differences were observed in the composition of other membrane components between wild-type and Colonia myxoamoebae, these results strongly suggest the involvement of poriferasterol monoglucoside in the active transport of D-glucose. Then we discussed that this substance may assist or regulate the action of glucose transporter protein in plasma membrane (20).
This substance may also be considered to act as an accelerator on the fusion of plasma membrane because the fusion of plasma membranes of mutant myxoamoebae occurred when the temperature was reduced, but wild-type myxoamoebae could fuse only in the case of conjugation of the cells of different mating types.
The biological significance of heat induction of steryl glucoside has not yet been clarified, but this substance may have some important role(s) in the process of heat-induced differentiation. It may act at an early step in a signal transduction system to trigger stress-induced differentiation; for example, it may regulate the heat-receptor on the membrane or assist a movement of active molecules in the membrane to induce successive heat responses. Another possibility is that this substance by itself may act as a mediator in a signal transduction system of heat stress. Additional investigations are necessary to clarify the biological significance of a glycosylation of membrane sterol in heat response and cell differentiation.
Recently, we also found a heat-induced expression of steryl glycoside in human cultured cells, and the purification and characterization of this substance are now underway. Hence, this phenomenon is not specified in Physarum cells and might have some important role(s) in all organisms.
We are indebted to Dr. Ichiro Yahara of the Tokyo Metropolitan Institute of Medical Sciences and Dr. Kazuhiro Nagata of Kyoto University for their encouragement and valuable discussion on this study.