Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA1
MUCAB, Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia2
Author for correspondence: Stephen Alexander. Tel: +1 573 882 6670. Fax: +1 573 882 0123. e-mail: alexanderst{at}missouri.edu
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ABSTRACT |
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Keywords: protein targeting, prespore vesicle (PSV), extracellular matrix (ECM), glycoproteins, plant cell wall
Abbreviations: ECM, extracellular matrix; LPS, lower pad solution; PSV, prespore vesicle
a Present address: Proteome Systems Limited, Locked Bag 2073, North Ryde Sydney, NSW 1670, Australia.
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INTRODUCTION |
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The spore coat of the mature spores is a tri-lamellar, polarized structure and has been studied by electron microscopic as well as biochemical methods. The outer layer of the spore coat is composed of loosely associated proteins, the middle layer contains cellulose and the inner layer of the spore coat is composed of proteins that are bound covalently to the coat (Devine et al., 1982 ; Hemmes et al., 1972
; Orlowski & Loomis, 1979
). A layer of galactose-N-acetylgalactosamine-containing mucopolysaccharide (GPS) lies just proximal to the plasma membrane of the underlying dormant amoeba (Erdos & West, 1989
). A multiprotein complex called the PsB complex is found within the spore coat of mature spores and spans the three layers of the spore coat. The PsB complex is made up of four major glycoproteins, SP96, PsB/SP85, SP70 and SP60, which are held together by both covalent and non-covalent bonds (Watson et al., 1993
). The proteins of the complex are expressed during the slug stage of development and accumulate in this preassembled form in prespore vesicles (PSVs) (Devine et al., 1983
; Watson et al., 1994
). At the final stage of spore differentiation, an unknown developmental signal induces the synchronous fusion of the PSVs with the plasma membrane followed by the subsequent secretion of the PsB complex, as well as other uncomplexed spore coat proteins which are stored in the PSVs. One such glycoprotein is SP75, which is not part of the preassembled PsB complex in the PSVs, but is subsequently assembled into the spore coat upon secretion (Watson et al., 1994
). Analysis by immunofluorescent staining with mAbs has shown that the PsB glycoprotein complex becomes part of both the spore coat and the interspore matrix which can be visualized as a phase-dense material between mature spores (Watson et al., 1994
).
The PsB complex has an endogenous cellulose-binding activity, a function which correlates well with its localization to the cellulose-rich spore coats (McGuire, 1996 ; McGuire & Alexander, 1996
). Using a series of mutants lacking different proteins of the PsB complex (Fosnaugh et al., 1994
), we established the relative order of assembly of the proteins into the complex and showed that a fully assembled PsB complex is required for cellulose-binding activity (McGuire & Alexander, 1996
). Specifically, we found that SP96 was anchored to the complex through SP70, which in turn was bound to PsB. The abrogation of either SP96 or SP70 resulted in the assembly of partial complexes (containing either SP70/PsB/SP60 or SP96/PsB/SP60, respectively) which did not have cellulose-binding activity. SP60 is anchored to the complex through PsB, but interestingly, its absence has no effect on the cellulose-binding activity of the partial complex containing SP96/SP70/PsB.
The cellulose-binding activity of the PsB complex suggested that this activity may be necessary for localization and proper assembly into the spore coat. Thus, we predicted that mutants which are defective in the cellulose-binding activity (SP96- and SP70-) will have an aberrant spore coat. To test this hypothesis, we examined the mutants for (1) spore coat protein localization, (2) spore coat structure and (3) viability of the mutant spores. The availability of several well characterized mAbs to the peptide and oligosaccharide epitopes of the different proteins of the PsB complex has enabled us to address these questions (Champion et al., 1991 , 1995
; Smith et al., 1989
).
In this report, using a combination of immunofluorescence microscopy and biochemical methods, we show that in the cotA- (SP96-) and cotB- (SP70-) mutant strains, the partial PsB complexes which lack cellulose-binding activity are secreted and do localize to the spore coat. This suggests that assembly of all components of the complex and the associated cellulose-binding property are not a prerequisite for its routing, secretion and localization. However, in these mutants, the structure of the spore coat is altered and results in a dramatic loss of viability of the spores compared to their wild-type parent. On the other hand, the cotC- (SP60-) mutant in which the partial PsB complex retains its cellulose-binding activity, produces spores with apparently normal structure. Thus the proper construction and assembly of the spore coat and the viability of the spores is directly correlated with the cellulose-binding activity of the PsB multiprotein complex.
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METHODS |
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Spore viability assays.
Strains were mass-plated in association with Klebsiella aerogenes. Spores of each strain were tested for viability by two methods, according to a procedure adapted from Richardson & Loomis (1992) . In the first test, spores were allowed to remain on stalks on slime mould medium plates at 22 °C for the duration of the experiment (30 d). Spores were collected every 5 d, resuspended in 10 mM EDTA at 1x105 ml-1 and incubated at 37 °C for 30 min. Duplicate serial dilutions in Bonners standard solution/5 mM EDTA were plated on slime mould medium plates in association with K. aerogenes. The number of plaques formed was counted and spore viability was expressed as the percentage of the number of plaques seen on day 1 of the experiment. In the second test, on day 1 spores were collected and resuspended to 1x108 ml-1 in 50 mM sodium phosphate, pH 6·4, with 1% antibiotic/antimycotic (Gibco-BRL). The spore suspensions were then shaken at 22 °C for the duration of the experiment (30 d). Viability was tested every 5 d as described above. Note that for the cotABC- strain, owing to a severe loss in viability, it was not possible to conduct the experiment for longer than 20 d.
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RESULTS |
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cotA deletion mutant (SP96).
The cotA- strain lacks the SP96 protein. As expected, no labelling with MUD3 was observed with either untreated (Fig. 1B, c
) or germinated spores (Fig. 1B
, e
). In contrast to the wild-type parental strain, some MUD102 fluorescence was visible in intact, untreated spores (Fig. 1B
, g
) and stronger MUD102 fluorescence was seen in germinated spores (Fig. 1B
, i
). These data indicate that the partial PsB complex still localizes to the spore coat in the absence of SP96. However, the PsB protein, which normally localizes to the inner layer of the coat is now exposed and available for recognition by MUD102 (Fig. 4b
).
cotB deletion mutant (SP70).
The cotB- strain lacks the SP70 protein. Although there are no mAbs available that specifically recognize this protein, the fate of other coat proteins can be followed in this strain. Previously, it has been shown that this protein mediates the association of SP96 (cotA) with the PsB protein because in cells lacking SP70, SP96 is no longer associated with the complex (McGuire & Alexander, 1996 ). When cotB- spores were stained with MUD3, strong fluorescence could be seen on the surface of both ungerminated (Fig. 1C
, c
) and germinated spores (Fig. 1C
, e
). These results indicate that SP96 is localized to the spore coat even when not in association with the rest of the PsB complex prior to secretion. The spores labelled with the MUD102 fluoresced brightly, in both intact (Fig. 1C
, g
) and germinated spores (Fig. 1C
, i
), demonstrating a clearly altered spore coat structure in mutants lacking SP70 where PsB is now available for binding to MUD102 in intact spores (Fig. 4c
).
cotC deletion mutant (SP60).
The cotC- strain lacks the SP60 protein. The absence of this protein does not affect the assembly of the rest of the PsB complex or its cellulose-binding activity (McGuire & Alexander, 1996 ). As expected, MUD3 fluorescence was observed in both the untreated (Fig. 1D
, c
) and germinated spores (Fig. 1D
, e
), indicating that the localization of SP96 was not affected by the loss of SP60. MUD102 labelling was not visible on intact spores (Fig. 1D
, g
) but was bright on germinated spores (Fig. 1D
, i
), indicating that spores lacking SP60 have a normal spore coat structure (Fig. 4d
). Interestingly, ungerminated immature spores in this sample did show some fluorescence with MUD102 (Fig. 1D
, g
), presumably because the coat is not yet completely matured, and suggests that development in this strain may be somewhat slower than the wild-type.
cotABC deletion mutant (SP96, SP70 and SP60).
The cotABC- strain lacks the SP96, SP70 and SP60 proteins. When spores from this strain were labelled with MUD3, no fluorescent signal was seen on either untreated (Fig. 1E, c
) or germinated spores (Fig. 1E
, e
). The MUD102 fluorescence was very bright on intact spores (Fig. 1E
, g
), though somewhat less bright on germinated spores (Fig. 1E
, i
). This labelling confirms that the PsB protein is still present and localized to the spore coat, but that the structure of the spore coat is greatly altered (compare Figs 1A
, g
and 1E
, g
).
Flow cytometry was performed on unfixed spores to confirm the results obtained from the immunofluorescence experiments, thus eliminating the possibility of fixation artefacts. MUD3 staining showed that SP96 was present on the outer layer of the spore coat in wild-type, cotB- and cotC- spores and, as expected, was absent in spores from cotA- and cotABC- strains. PsB was exposed to the outside of the spores in the cotA-, cotB- and cotABC- strains, but not in the wild-type and cotC- spores (data not shown).
Biochemical extractions.
The immunofluorescent analysis showed that the structure of the spore coat was altered in mutants with partial PSB complexes which lack cellulose-binding activity. To determine whether these changes in coat structure were reflected at the biochemical level, we examined the structure of the spore coat in the parental strain HL328 and the four cot mutant strains by a series of extractions. The extracted proteins were separated by SDS-PAGE and analysed by Western blotting, using the mAbs MUD62, MUD141, MUD3 (all recognizing SP96) and MUD102 (recognizing PsB) (Table 2 and Fig. 2
). The biochemical data confirm the results from immunofluorescence experiments and show that spores from the cotA-, cotB- and cotABC- strains are altered, and that the spores from the cotC- strain are similar to the wild-type.
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It is important to note that the majority of SP96 (wash 3) does not co-fractionate with the majority of the PsB protein (wash 4), although SP96 has been shown to be covalently linked to the PsB complex through disulfide bridges (Watson et al., 1993 ). This observation suggests that SP96 is expressed in large excess over the other proteins in the PsB complex. This idea is supported by experiments in which labelled cell extracts that were depleted of the PsB multiprotein complex by sequential immunoprecipitations with MUD102 still contained free SP96, as demonstrated by immunoprecipitation with MUD62 (unpublished).
cotA deletion mutant (SP96).
SP96, assayed by MUD62, MUD3 or MUD141, is missing from all the fractions (Fig. 2b). PsB was found only in fraction 4 from this strain, confirming that in the absence of SP96, the partial PsB complex can still localize and be covalently bound to the spore coat. However, the distribution of PsB to the interspore matrix appears to depend on the presence of SP96.
cotB deletion mutant (SP70).
In the absence of SP70, SP96 does not associate with the PsB complex (McGuire & Alexander, 1996 ). The immunofluorescence data indicated that SP96 is still incorporated into the spore coat in this mutant. However, staining with MUD141 and MUD3 show that the distribution of SP96 is altered in this mutant (Fig. 2c
). There was an increase in SP96 in fractions 1 and 2 in contrast to the parental strain HL328 (compare Fig. 2a
and 2c
), because when SP70 is absent, SP96 is not bound to the PsB complex and therefore is extracted under milder conditions (wash 1). The PsB distribution was virtually identical to that observed in the parental strain.
cotC deletion mutant (SP60).
The distribution of SP96 in this strain is identical to the parental strain with almost all of SP96 in fraction 3 (Fig. 2d). The pattern of MUD102 labelling was also identical to the parental strain.
cotABC deletion mutant.
The results with the triple mutant are identical to those seen with the cotA- strain, which also lacks SP96 (Fig. 2e). Again, only fraction 4 contains the PsB protein.
The SP75 glycoprotein is found in the spore coat of mature spores, but is not a component of the preassembled PsB complex (Watson et al., 1994 ; West et al., 1996
). It is recognized by the mAb MUD62 and in all strains tested, SP75 is extracted predominantly in the fourth wash (Fig. 2ae
). It is therefore cross-linked normally into the coat in the different cot mutants.
Spores with partial PsB complexes have lower viability
The immunofluorescence and biochemical extraction experiments described so far demonstrate a change in the structure of the spore coat in the cotA-, cotB- and cotABC- strains. No alteration was seen with the cotC- strain when compared to the parental strain. These data are in accord with our observation that only the cotC- strain produces a partial PsB multiprotein complex that still retains cellulose-binding activity (McGuire & Alexander, 1996 ). Since proper spore coat assembly and structure are crucial to the survival of the organism, we predicted that there would be a loss of spore viability in mutants where the spore coats were altered.
To test this, spores from the wild-type parent and the four mutant strains were tested for their viability over a period of 30 d. In the first test, spores were allowed to remain on stalks at 22 °C for the duration of the experiment and their viability was tested every 5 d as described in Methods. The data are presented in Fig. 3(a). The wild-type parent showed no loss of viability over 30 d. The most dramatic effects were seen in the cotA- and cotABC- strains, where a 1300- and 3750-fold loss in viability was observed, respectively. The cotB- strain showed an 18-fold reduction in viability. However, only a threefold decrease in viability was seen in the cotC- mutant spores.
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DISCUSSION |
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Using immunofluorescence microscopy and biochemical extractions, we first showed that partial PsB complexes and free SP96 do localize to the coat even when they do not have cellulose-binding activity. However, in the cases of the cotA-, cotB- and cotABC- strains, the coats are improperly assembled. The aberrant antibody staining of the mutant spores is due to an increased porosity of the spore coat or incorrect orientation of the PsB complex within the coat, or more likely a combination of the two factors. In contrast, deletion of the SP60 protein alone in the cotC- strain does not alter the structure of the coat (Fig. 4). Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure.
Examination of spore viability confirmed the correlation between cellulose-binding activity and normal spore coat assembly and provided a quantitative measure of the effect. Although loss of SP60 results in no apparent difference in spore coat structure, a small but measurable reduction in viability was observed (310-fold). This indicates that SP60 is an important structural component of the spore coat whose presence is essential for normal cell survival, even though it is not required for the cellulose-binding activity of the PsB complex. Our results are in disagreement with findings reported by Fosnaugh et al. (1994) who claimed that there was no compromised viability of spores from any of the spore coat protein mutants, although they did show an increased permeability to the lectin ricin A.
It has recently been shown that the C-terminal half of recombinant PsB protein binds cellulose in vitro (Zhang et al., 1998 , 1999
). The present study strongly supports our earlier work showing that PsB complexes that lack SP96 or SP70, but that do have the PsB protein, do not bind cellulose (McGuire & Alexander, 1996
). Thus, we suggest that the proper positioning of PsB within the fully assembled multiprotein complex allows the correct exposure of the PsB protein cellulose-binding site to cellulose in situ. In mutant strains lacking either the SP96 or SP70 proteins, the cellulose-binding site of PsB would not be available. Presumably, this idea can be tested when all the PsB complex proteins are expressed in vitro, although it must be remembered that each of these proteins is heavily glycosylated with specific oligosaccharides (Champion et al., 1995
).
Overall, this and earlier studies present a picture where assembly of the PsB complex proteins results in a multiprotein complex with a specific cellulose-binding activity. Neither the complete assembly of the complex, nor the resulting binding activity is required for secretion and localization into the spore coat. However, the cellulose-binding activity is required for proper assembly and organization of the spore coat, and for the subsequent viability of the spores. Under natural conditions this reduction in viability would dramatically affect their survival. We suggest that the primary function for the cellulose-binding activity of the PsB complex is to organize the cellulose fibrils so that the spore coat is impermeable and resistant to environmental stress (Fig. 4). Other proteins, such as SP60 and SpiA (Richardson & Loomis, 1992
), which do not affect cellulose binding, also play important structural roles in the formation and maintenance of the spore coat ECM.
Nothing is known about how cellulose synthesis is co-ordinated with its deposition in the spore coat at terminal morphogenesis. The cellulose synthase gene in D. discoideum has recently been cloned and is expected to reveal information about cellulose deposition and assembly, although its localization within the cell is not yet known (Blanton et al., 2000 ). It remains to be shown how the timing of cellulose synthesis in the prespore cells is co-ordinated with the secretion of the contents of the PSVs. Earlier work from West & Erdos (1990)
suggests that spore coat protein secretion occurs before cellulose deposition and this agrees well with the conclusions of this study, which shows that the cellulose-binding activity of the PsB complex is required for the normal assembly of the spore coat.
The structure of the D. discoideum spore coat is reminiscent of plant cell walls, in that both are complex matrices composed of glycoproteins, polysaccharides and cellulose. The genesis of plant cell walls is intimately linked to the growth and development of plant cells and tissues (Delmer, 1999 ). Glycoproteins from cell walls of angiosperms can also be extracted under different conditions. Loosely associated proteins can be washed off with buffer, proteins bound by ionic interactions can be removed by salt washes and covalently bound proteins are extracted under strong reducing conditions (McNeil et al., 1984
). During cell wall formation, cellulose is synthesized and extruded across the plasma membrane. The cellulose microfibrils are thought to self-assemble in the extracellular space, although the direction of cellulose deposition may be guided by microtubules lying adjacent to the membrane (Cosgrove, 1997
). Yet, the detailed mechanisms by which plant cell wall structures are assembled are largely unknown and it is possible that there are cellulose-binding proteins that play a role in guiding this process, similar to that occurring in D. discoideum spore coats.
Recently, the PSVs, which contain most of the components of the future spore coat have been purified from different stages of development (Srinivasan et al., 1999 ). This has allowed us to begin an exhaustive characterization of the PSV contents by proteomics (Yan et al., 1997
). An analysis of the protein content of the PSVs will reveal the presence of additional structural proteins of the spore coat, as well as enzymes which may be required for coat assembly and formation, such as protein disulfide isomerases. Such analyses are expected to increase our understanding of how the D. discoideum spore coat is assembled with its precise polarity and organization at terminal differentiation.
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ACKNOWLEDGEMENTS |
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Received 23 December 1999;
revised 11 February 2000;
accepted 25 April 2000.