Expression of Poriferasterol Monoglucoside Associated with Differentiation of Physarum polycephalum*

A glycolipid which was expressed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum, has been examined. In the amoeboid stage, cells did not contain this glycolipid, but after conjugation of the haploid cells, this substance appeared and increased in its amount. From structural studies of the purified glycolipid, it has been identified as poriferasterol monoglucoside.

In the life cycle of a true slime mold, Physarum polycephnlum, it has haploid and diploid stages (1)(2)(3). Haploid myxoamoebae are germinated from spores under moist conditions, and they conjugate in pairs to form diploid plasmodia. At the amoeboid stage, cells recognize different mating types and fuse to form zygotes. Zygotes grow and fuse each other and differentiate into young plasmodia. These steps are genetically regulated; zygote formation occurs only between amoebae carrying different alleles of a multiallelic locus (4)(5)(6)(7)(8) , and differentiation of the zygotes to plasmodia is also genetically determined (4,9, 10). Tiny young plasmodia grow up as multinuclear plasmodia, and they fuse each other without any artificial treatments. During the course of differentiation from haploid amoebae to diploid plasmodia, the change of membrane characteristics might occur.
We examined lipid composition of the membranes of the cells at both stages and found an expression of a novel glycolipid which was correlated with a process of differentiation. This glycolipid was purified and characterized and was identified as poriferasterol monoglucoside.

EXPERIMENTAL PROCEDURES AND RESULTS'
Expression of a Glycolipid during the Differentiation of P. polycephulum from Haploid Amoebae to Diploid Plasmodia-TLC analysis of crude lipid fractions (see "Experimental * 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.
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Procedures") showed the pattern of nonpolar glycoconjugates of haploid cells differed from that of diploid cells. Fig. 1 shows the TLC patterns of the nonpolar glycoconjugates from haploid amoebae, strain J, or diploid plasmodia of P. polycephalum. There was no difference between two different mating types of myxoamoebae, strains J and F. Four major carbohydrate-containing bands from haploid cells were designated as A to D according to their mobility. Five major sugar-containing bands were obtained in the extract from diploid cells: three of them were corresponding to A, C, and D from haploid cells, and other two bands designated as E and F were characteristics for diploid stage. Band D showed blue color and others showed purple colors.
Among these nonpolar glycoconjugates, substance F was expressed after conjugation of haploid amoebae and increased in its amount during the differentiation into diploid plasmodia (Fig. 2). A content of this substance was maintained constantly under different culture conditions either on rolled oats or in semidefined culture media. Substance E appeared after the differentiated plasmodia were transferred into culture media. When the crude membrane fraction was extracted, bands A and B were not detected, but other glycoconjugates were also extracted and almost the same results were obtained.
Purification and structural analysis of glycolipid F are presented in the Miniprint Supplement.
From the evidence described in the Miniprint Supplement, the structure of substance F was designated as that shown in

DISCUSSION
In this work it is shown that a novel glycolipid was expressed during a differentiation of haploid amoebae of P.
polycephalum into diploid plasmodia. The glycolipid was purified and identified as poriferasterol monoglucoside.
Poriferasterol was a major sterol component in haploid cells of P. polycephalum (34) as well as in diploid cells (25). But in a haploid stage, the poriferasterol monoglucoside was completely absent, and during the differentiation into diploid plasmodia it appeared and increased in its amount. Other steryl-glucosides and their derivatives could not be detected in both stages.
The steryl-monoglucosides and their 6"O-acyl derivatives are known as common constituents of higher plants (27,28), and their biological functions have been suggested to be metabolically active components of plant membrane structure (291, intercellular transporters of sterols (30), or glucose carriers through cell membranes (31, 32).
Amoeboid cells are uni-nuclear and behave like protozoan soil-amoebae on solid substrata or amoebo-flagellate in non- nutrient liquid. Diploid cells grow as multinuclear plasmodia in which an intranuclear mitosis occurs, and they fuse each other very easily without any artificial treatments. The cell membrane of plasmodia seems to have higher fluidity than that of haploid amoebae, and when the cell membrane has been injured the repair of it is completed immediately. Plasmodia are capable of growth in liquid or on agar media, but amoebae, except for rare mutant strains (33), can be cultured only on bacterial lawns. Amoebae may not be able to utilize glucose and other small molecules, but plasmodia may be capable of utilizing them as nutrients. Poriferasterol monoglucoside may have some active functions in membranes showing such interesting properties. It seems that the role of poriferasterol monoglucoside in the plasmodial membrane must be examined with purified membrane components and compared to properties of the amoeboid membrane.
It is also important to examine the regulation of the enzyme, UDP-g1ucose:poriferasterol glucosyltransferase, which synthesizes substance F, during the process of differentiation of P. polycephalum.

Differentiation of Physarum
16723 sterol or porlferasterol, latter one 1s a C-24 eplmer Of former one. The 400 MHz NMR spectra Of the sterol molety of the Substance F showed rt n o t to be a stlgmasterO1 but a Porlferasterol, ludglng from the methyl group chemlcal shifts I F q . 9 1 whlch were reported by Rublnsteln ef a 1 . ( 2 6 1 before. The melti n g p o m t of It was 1 5 6 -C and thls v a l u e also conflrmed that the sterol was not a stlgrnasterol (m.p., 170'Cl but a porlferasteral 1m.p.. 1 5 6 -C J as described by Bullock and Dawson ( 2 5 1 .
From above results, the sterol molety could not be ldentrtied a s stlgrna-