PsB Multiprotein Complex of Dictyostelium discoideum
DEMONSTRATION OF CELLULOSE BINDING ACTIVITY AND ORDER OF PROTEIN SUBUNIT ASSEMBLY*
- § Recipient of American Cancer Society Faculty Research Award FRA448. To whom correspondence should be addressed. Tel.: 573-882-6670; Fax: 573-882-0123; E-mail: salex{at}biosci.mbp.missouri.edu
Abstract
The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.
Footnotes
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↵‡ Presented in partial fulfillment of the requirements for the Ph.D. degree from the University of Missouri.
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↵* The work was supported by grants from the National Science Foundation (IBN9206891) and the University of Missouri Research Board. The costs of publication of this article were defrayed in part by the payment of page charges. The 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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↵1 The abbreviations used are:
- ECM
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extracellular matrix
- AEBSF
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4-(2-aminoethyl)benzenesulfonyl fluoride
- mAb
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monoclonal antibody
- PsA
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prespore protein A
- PsB
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prespore protein B
- PSVs
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prespore vesicles
- PAGE
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polyacrylamide gel electrophoresis
- CBD
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cellulose binding domain.
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↵2 The PsB complex proteins were defined by their molecular weights on reducing SDS-PAGE. The complex includes four major proteins, p112, PsB (p100), p78, and p58 and two minor proteins, p70 and p63 (Watson et al., 1993). The detection of the two minor species is dependent on a longer period of radiolabeling indicating they are synthesized at a lower rate. In this study, we have used short periods of labeling and these two proteins are not obvious and are not considered in this analysis. Subsequent work (Watson et al., 1994) identified p112 as the major phosphorylated spore coat protein SP96 identified by Loomis et al. (Devine et al., 1983). We also showed that the antibody mAb16.2 (which is reactive with a protein called SP85 (West et al., 1986)) recognizes PsB.
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↵3 V. McGuire and S. Alexander, unpublished observations.
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- Received February 1, 1996.
- Revision received April 4, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
