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Supplementary Figure Legends
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Figure S1. Tryptic peptide sequence alignment, peptide synthesis, and dot-blot assessment
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of peptide antibodies raised against COS bacterial-type PEPC. (a) ClustalX was used to
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align the sequence of a tryptic peptide derived from the p64 subunit of purified Class-2 PEPC
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from developing COS (Blonde and Plaxton, 2003) with the corresponding region of several
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bacterial- and plant-type PEPCs. A 12 amino-acid sequence (dotted box in panel a, underline
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in panel b) was selected for peptide synthesis and subsequent antibody production. Identical
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and similar amino-acids are indicated by black and light grey shading, respectively. The
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abbreviated name of each aligned sequence is as follows: AtPPC1–AtPPC4, A. thaliana
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Ppc1–Ppc4; GmPPC7 and GmPPC17, Glycine max Ppc7 and Ppc17, respectively; OsPPC1
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and OsPPC-b, Oryza sativa Ppc1 and Ppc-b, respectively. Corresponding NCBI protein
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accession numbers are listed in Experimental procedures. (b) Sequence of synthetic peptide
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that was covalently coupled to keyhole limpet hemocyanin and used for rabbit immunization.
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(c) Dot blots of varying amounts of the synthetic peptide (b) were probed with several
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dilutions of the affinity-purified anti-(COS BTPC)-IgG in the absence or presence of 10 µg/mL
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of the blocking parent peptide. Immunoreactive polypeptides were visualized as described in
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the Experimental procedures.
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Figure S2. Immunoblot and/or SDS-PAGE analysis of recombinant AtPPC4-CT, AtPPC4,
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and AtPPC3. Open reading frames encoding a 15-kDa polypeptide corresponding to the C-
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terminal (CT) end of AtPPC4 (a-c), as well as 116- and 110-kDa polypeptides corresponding
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to full-length AtPPC4 and AtPPC3, respectively (d), were over-expressed in E. coli as
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described in Appendix S2. SDS-PAGE was followed by protein staining (a, b, d) or
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immunoblotting (c). (a) Lanes 1 and 2 respectively represent the soluble and insoluble
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fractions of the AtPpc4-CT expressing BL21 CPRP E. coli strain, while lanes 3 and 4
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represent the corresponding soluble and insoluble fractions of the control pLySs E. coli strain
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containing an empty pET29a vector (15 µg protein/lane). The 15-kDa AtPPC4-CT
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polypeptide (lane 2) was excised from a SDS-PAGE preparative gel, electroeluted, and
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dialyzed against PBS for rabbit immunization. The electroeluted, dialyzed 15-kDa
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polypeptide was analyzed by SDS-PAGE (5 µg protein) (b), or immunoblotting (50 ng
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protein) (c) with 100-fold diluted affinity-purified anti-(AtPPC4-CT)-IgG. (d) Lanes 1 and 2
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respectively represent the soluble and insoluble fractions of the AtPpc4 expressing BL21
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CPRP E. coli strain, lanes 3 and 4 represent the soluble and insoluble fractions of the AtPpc3
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expressing BL21 CPRP E. coli strain, while lanes 5 and 6 represent the corresponding
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soluble and insoluble fractions of the control pLySs E. coli strain containing an empty
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pET29a vector (25 µg protein/lane).
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Figure S3. Mono-Q HR5/5 elution profile of COS p90. This was the last step of p90
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purification using an ÄKTA FPLC system as described in Appendix S1. Inset: Immunoblot
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analysis of various fractions (5 µl each) probed with affinity-purified anti-(COS BTPC)-IgG as
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described in the legend for Figure 2.
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Figure S4. SDS-PAGE and immunoblot analysis of p90 (sucrose synthase) isolated from
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stage VII developing COS. (a) SDS-PAGE (10% separating gel) of purified p90. The lane
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labeled ‘M’ contains 2.5 µg of various protein standards. (b) Immunoblot analysis was
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performed using a 1:50,000 dilution of anti-(soybean root nodule sucrose synthase)-immune
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serum. Antigenic polypeptides were visualized as described in the Experimental procedures.
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Figure S5. Influence of pH, COS development, and various protease inhibitors on in vitro
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proteolysis of the p118 BTPC during incubation of clarified COS lysates on ice. Aliquots were
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removed at the indicated times and subjected to immunoblot analysis using anti-(COS
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BTPC)-IgG. (a) Clarified stage VII COS extracts at the indicated pH were incubated for up to
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48 h on ice in extraction buffer lacking HEPES but containing 50 mM Bis-Tris-Propane, 20%
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ammonium sulfate (AS), & 2 mM DTT. (c) Extracts from endosperm harvested at various
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stages of COS development were incubated at pH 7.5 on ice in the presence of 20% AS and
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2 mM DTT. Developmental stages are as described in the legend for Figure 2. (d) Influence
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of protease inhibitors on the proteolytic susceptibility of p118 in clarified extracts from stage
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VII developing COS. Extracts (pH 7.5) were incubated on ice in the presence of 20% AS with
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various additions as indicated. ‘PMSF’ denotes 10 µl/mL of G-Biosciences’ PMSF stock
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solution (Product #786-207), ‘Sigma PIC’ denotes 7 µl/mL of Sigma’s Plant Protease Inhibitor
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Cocktail (Product #P-9599), ‘Roche PIC’ denotes 1 tablet/50-mL of Roche’s Protease
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Inhibitor Cocktail Tablets (Product #11-697-498-001), ‘Calbio. PIC’ denotes 17 µl/mL of
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Calbiochem’s Plant Protease Inhibitor Cocktail (Set VI, Product #539-133), ‘ProteCEASE
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PIC’ denotes 10 µl/mL of G-Biosciences’ ProteCEASE 100 Protease Inhibitor Cocktail
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(Product #786-336), ‘PPA PIC’ denotes 10 µl/mL of G-Biosciences’ Plant Protease Arrest
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Inhibitor Cocktail (Product #786-332). Note: The following substances exerted little to no
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inhibition of p118 proteolysis as judged by immunoblotting of COS extracts incubated for up
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to 21 h (results not shown): EDTA and EGTA (15 mM each); 1x concentrations of all the
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following compounds from G-Biosciences’ Protease Inhibitor Kit (Product #786-207),
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comprised of separate 100x stocks containing unspecified concentrations of aprotinin,
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bestatin,
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hydrochloride); leupeptin or pepstatin (25 µg/mL each).
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phosphoramidon,
antipain,
and
4-(2-aminoethyl)
benzenesulfonyl
fluoride