Heat Stress Induces a Glycosylation of Membrane Sterol in Myxoamoebae of a True Slime Mold, Physarum polycephalum *

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 glyco- lipid 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. Wojciechowski et al . (9, 10) with slight modification (2). Other Methods— Colorimetric determination of hexose and sterol was performed according to Radin et al . (11) and Momose et al . (12), respectively.

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 heatshock 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.

EXPERIMENTAL PROCEDURES
Organisms-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.
Lipid Extraction and Purification of a Glycolipid-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).
TLC-Lipids were analyzed by TLC in solvent system I and II, and glycolipids were visualized by spraying with 2% orcinol in 2 N H 2 SO 4 followed by heating at 125°C. Other color-developing reagents, such as resorcinol-HCl for sialic acid, azure A-H 2 SO 4 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 phosphatecontaining 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 Spectrometry-Fast atom bombardment mass spectrometry for an intact glycolipid was carried out on a JEOL DX-303 mass spectrometer under the described conditions (1).
Assay of UDP-glucose:Poriferasterol Glucosyltransferase-The assay of this transferase was performed essentially by the procedure of * This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan and from Salt Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Other Methods-Colorimetric determination of hexose and sterol was performed according to Radin et al. (11) and Momose et al. (12), respectively.

RESULTS
Expression of a Glycolipid in the Heat-shocked Myxoamoebae-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 heatshock 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-X-GL-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-H 2 SO 4 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] Ϫ 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 R F 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).
Induction of UDP-glucose:Poriferasterol Glucosyltransferase Activity in Heat-shocked Myxoamoebae-Endogenous UDPglucose: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.

DISCUSSION
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 UDPglucose: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 6Ј-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 with-   out 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.