Plant Physiol. (1986) 80, 950-9550032-0889/86/80/095o/o6/$o 1.00/0

Glycoproteins in the Matrix of Glyoxysomes in Endosperm ofCastor Bean Seedlings'

Received for publication October 1, 1985 and in revised form December 10, 1985

ELMA GONZALEZDepartment ofBiology, University ofCalifornia,

ABSTRACT

The matrix of glyoxysomes from endosperm of castor bean (Ricinuscommunis cv Hale) seedlings has been analyzed for the presence ofglycosylated proteins. Glyoxysome preparations were monitored for or-ganelle homogeneity by electron microscopy and enzyme marker activi-ties. Glyoxysomes were essentially free of endoplasmic reticulum, mito-chondria, and protein bodies. At least eight glyoxysomal matrix glyco-peptides ranging in size from 39 to 160 kilodaltons were identified bytheir affinity for concanavalin A. The glyoxysomal glycoproteins wereshown to be radioactively labeled when endosperm was allowed to incor-porate glucosamine. Incorporation of glucosamine was inhibited by tuni-camycin under conditions which did not inhibit protein synthesis. Hy-drolysis of glyoxysomal extracts and subsequent analysis by paper chro-matography showed that the labeled precursor was incorporated into theglycoprotein without prior dispersion of the label into amino acids. Thepresent data demonstrate the occurrence of N-linked, high mannoseoligosaccharides on polypeptides of the glyoxysomal matrix. This findingis discussed in relation to pathways of protein maturation and transportduring glyoxysomal biogenesis.

In castor bean endosperm, as in the storage tissues of other oilseeds, the onset of imbibition initiates a complex pattern ofprotein synthesis, membrane assembly, and organelle prolifera-tion. In these tissues a prominent and essential feature of seedlinggrowth is the biogenesis of glyoxysomes and the synthesis ofglyoxysomal enzymes. Imbibition is known to trigger transcrip-tion and accumulation of mRNAs specific for glyoxysomal en-zymes, e.g. isocitrate lyase (31). Isocitrate lyase is synthesized onnonmembrane bound polysomes (39) as are other glyoxysomalenzymes (14, 23).The detailed features of the subcellular pathway of the glyox-

ysomal proteins from their site of synthesis on free polysomes totheir eventual sequestration in the mature glyoxysome are notcompletely understood. There are currently two models proposedfor intracellular transport of the glyoxysomal matrix proteins.The first of these, developed primarily from work on cucurbitcotyledons, suggests that the path of proteins from the cytosolinvolves an oligomeric precursor pool (23, 24). Proteins are thenthought to be sequestered into preformed glyoxysomes (22). Thesecond hypothesis, based almost entirely on work on castor beanendosperm, suggests that the ER serves as staging area for accre-tion and possible modification of glyoxysomal matrix enzymes;vesiculation of specialized ER domains then leads, eventually, tomature glyoxysomes (2).

Various lines of evidence support the conclusion that the

'Supported by National Science Foundation PCM 82-04539.

Los Angeles, Los Angeles, California 90024

glyoxysomal membrane is derived from the ER in castor bean(reviewed in Beevers [2]). The role of the ER as a staging site forglyoxysomal matrix proteins, however, is still under debate (40).Although it has been shown that enzymes unique to the glyoxy-some are associated with the ER at times of rapid glyoxysomalproliferation (15-17, 21, 26), there has been no convincingdemonstration of direct participation of the ER in the posttrans-lational processing ofglyoxysomal proteins. One type of process-ing known to occur in ER of endosperm is glycosylation.The present study surveys the entire complement of glyoxy-

somal matrix proteins for the presence ofglycoproteins. Evidenceis presented that demonstrates the existence of a number ofglycoproteins in the glyoxysomal matrix.

MATERIALS AND METHODS

Tissue Preparation. Seeds of castor bean (Ricinus communis,cv Hale) were planted in vermiculite as described previously (17).Two-d germinated seedlings wvere treated with 100 .M GA3 (13)for 12 to 16 h prior to exposure to radioactive label. Tissue (8-10 endosperm halves) was minced with a razor blade in 4 ml ofgrinding medium consisting of 20% (w/w) sucrose; 100 mmTricine (pH 7.5); 10 mM KCI; I mm EDTA; and 2 mM MgC12.The homogenate was filtered through Miracloth as described(13).

Isolation of Glyoxysomes and ER. Glyoxysomes and ER wereisolated in sucrose gradients. A continuous, concave gradientwas achieved by forming two sequential, linear gradients in thesame tube, i.e. 4 + 4 ml, 40 to 20% (w/w), and 2 + 2 ml, 60 to40% (w/w) sucrose with 1 ml 60% sucrose as an underlyingcushion. Gradient solutions contained sucrose, 100 mm Tricine,10 mm KCI, and I mM EDTA. Loaded gradients were subjectedto centrifugation in the Beckman SW 27.1 rotor (20,000 rpm for3 h; w2t = 4.74 x 10'°). For larger preparations, gradients werescaled appropriately and centrifuged in the Beckman VTi 50rotor (25,000 rpm for 32 min; w2t = 1.32 x 10'0) in a BeckmanL8-70M Ultracentrifuge. Gradients were fractionated (0.44 or0.9 ml fractions) by an ISCO (Instrumentation Specialties Co.,Lincoln, NE) apparatus. In these gradients the slope of increasein sucrose concentration at the top of the tube is more gradualthan the slope at the bottom ofthe tube. This simple modificationallows complete separation ofthe ER (which equilibratesbetween28 and 31% sucrose) from the soluble, cytosolic componentswhich remain at the top of the tube.

Pooled ER (1.14 g/cm3) or glyoxysomal (1.24 g/cm3) fractionswere diluted with water and KCI to yield final concentrations of18% sucrose and 0.2 M KCI. After stirrjng for 15 min themembranes were removed by centrifugation (Beckman 5OTirotor at 45,000 rpm for 45 min; W2t = 5.84 x 10'0). Thisprocedure solubilizes most but not all of the protein peripherallyassociated with the membranes. For the purposes of this studyall proteins removable by KCI are treated as components of thematrix.

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GLYOXYSOMAL MATRIX GLYCOPROTEINS

Label Application. Labeled compounds were applied to theadaxial surface of the endosperm halves or to 1 mm tissue slices(for tunicamycin treatment). (D-[U-'4C]glucosamine hydrochlo-ride, 277 mCi/mmol) and (L-[35S]methionine, 1300 Ci/mmol)were obtained from Amersham (Arlington Heights, IL). N-Ace-tyl-D-[ 1 ,6-3H]glucosamine, 33 Ci/mmol was obtained from NewEngland Nuclear.

Immunoprecipitation. Glyoxysomes from 4-d-old endospermwere isolated on linear sucrose gradients. Matrix proteins wereobtained by osmotic shock (in 0.2 M KCI) of glyoxysomes andremoval of membranes by centrifugation. Antiserum was raisedin rabbits after repeated immunization with matrix proteins incomplete Freund's adjuvant. A 5-fold concentrated y globulinfraction was used for immunoprecipitation.

Immunoprecipitation was essentially as described (39). La-beled organelles were diluted 1:1 with buffer (10 mm Tris [pH7.6], 1% [v/v] Nonidet P-40, 2 mM EDTA, 150 mM NaCl, 40Ag/ml phenylmethylsulfonylfluoride), mixed vigourously andsubjected to centrifugation (7,000g for 10 min) to remove partic-ulate material. Antigens were precipitated using Protein A-Se-pharose (Pharmacia) and 7 jl of immune gamma globulin.Acrylamide Gels and Fluorography. Polyacrylamide slab gels

(10%) containing 0.05% SDS were cast as described (25). Elec-trophoresis was carried out under reducing conditions generallyfor 1.5 h at 0.3 mamp/cm followed by 4 h at 1.0 mamp/cm.Gels were prepared for fluorography as described (4). Dried gelswere applied to Kodak XRP-5 x-ray film and stored at -70°Cfor intervals ranging from 3 to 5 d.

Protein Transfer to Nitrocellulose Membranes. Protein trans-fer from SDS-acrylamide gels to nitrocellulose membranes (Bio-Rad, Trans Blot Transfer Medium No. 162) was carried out in alocally manufactured transfer apparatus having a distance be-tween electrodes of 6 cm. Electrotransfer was carried out at 24V (200 mamp) for 18 to 24 h in a transfer solution containing20 mm Tris-base, 150 mM glycine, 20% methanol, and 0.1% SDS(pH 7.8).

Lectin lodination and Glycoprotein Analysis. Con A2 (Sigma)was iodinated as described (29). Lectin overlay of protein blotswas carried out essentially as described by Burridge (6). Thenitrocellulose blot was exposed to a solution containing 3% BSA,10 mm Tris (pH 7.4), 0.9% NaCl, 0.5 mM CaCl2, and 0.5 mMMnCl2 for I h at 40°C. The blot was rinsed in distilled H20,placed in a heat seal bag, and allowed to react with a ['25I]ConA solution containing approximately 106 cpm/ml per 30 cm2 ofnitrocellulose and 0.3% BSA, buffer, and salts as above. Incu-bation was at 30°C for 18 h with gentle shaking. After rinsing inthe buffer and salts solution, the nitrocellulose paper was blottedto remove excess moisture, covered with plastic wrap, and placedon x-ray film for autoradiography for intervals ranging from 3to 5 d.

Electron Microscopy. Gradient fractions containing glyoxy-somes were pooled. Glutaraldehyde in 50% (w/w) sucrose wasadded to make 2% glutaraldehyde and the organelles fixed for62 h at 5°C. Fixed glyoxysomes were diluted to 38% (w/w)sucrose, sedimented, and washed in 50 mm K-phosphate (pH7.5) containing 50% (w/w) sucrose. Pellets were postfixed in 1%OS04 and 50% sucrose in 50 mM K-phosphate (pH 7.5) for 90min at 5°C. Samples were washed in buffer and embedded inagar. After dehydration in ethanol the pellets were embedded inMedcast plastic (Pellco).

RESULTSIsolation of Glyoxysomes. Linear sucrose gradients have been

used repeatedly to isolate intact and nearly homogenous popu-lations of organelles from castor bean endosperm (2, 3, 5, 8, 13-

18, 21, 26, 30, 32, 34, 37). The procedures described in thispaper are only minimally changed from those used by Huangand Beevers (20) and Donaldson and Beevers (9) who haveshown by marker enzyme analyses and electron microscopy thatrecovered glyoxysomes were nearly free of contamination byother membranous organelles. In more recent studies, usingmethods nearly identical to those reported here, E. Gonzmiez etal. (unpublished data) and T. K. Fang and R. P. Donaldson(unpublished data) have shown that ER preparations containedonly 1.6% of the glyoxysomal isocitrate lyase specific activityand 3.4% of the mitochondrial Cyt c oxidase specific activity.Similarly, glyoxysomal preparations contained 5.4% of the phos-phorylcholineglyceride transferase (ER marker) and 1.5% of themitochondrial Cyt c oxidase specific activity.

Electron microscopy of organelles in gradient fractions re-vealed a degree of homogeneity consistent with findings basedon enzyme marker analyses (Fig. 1). Electron micrographs takenat random from sections of glyoxysomal pellets (two separatepreparations) were examined for the presence of mitochondriaor other membranous organelles. Based on actual count oforganelle profiles, 5% could be identified as mitochondria, and3% as other (probably proplastids). Thus, at most, glyoxysomalpreparations are contaminated by 5% mitochondria, 5% ER,and 3% proplastids.Matrix Protein Affinity for Concanavalin A. Matrix proteins

were released, by osmotic shock in 0.2 M KCI, from the glyoxy-somal membrane and subjected to SDS-PAGE. The polypeptidebands were electroblotted from the gel onto nitrocellulose mem-branes. The polypeptides in the western blot were examined foraffinity for Con A in the presence or absence of a-methylgluco-side (Fig. 2). A least eight of the polypeptides in the resultantwestern blot reacted with ['25I]Con A in a specific manner, asjudged by a-methylglucoside competition. The glycoproteinsidentified ranged in mol wt from 39 to 160 kD.

Additional experiments were conducted to explore the possi-bility of contamination by protein body glycoproteins. Althoughprotein bodies have densities similar to glyoxysomes (1.24 g/cc),they do not survive the homogenization and fractionation con-ditions used in these studies. In fact, intact protein bodies werenot observed in electron micrographs of the glyoxysome prepa-ration (Fig. 1). Nevertheless, the possibility of contamination byprotein body components was explored further. Since approxi-mately 10% of protein body protein is ricin, a galactose-bindinglectin, contamination by protein body components would alsohave led to detectable amounts of ricin in the glyoxysomal matrixpreparation. To test this possibility, lactose-agarose beads wereadded to a portion of the glyoxysomal matrix preparation underconditions that favor ricin binding (SM Harley, personal com-munication). Proteins which did not bind to lac-agarose weresubjected to SDS-PAGE, western blotting, and Con A. Exposureto lactose-agarose did not reduce the number of polypeptidescapable of binding Con A (cf. lanes A and B in Fig. 3), therebydemonstrating the absence of ricin from the glyoxysomal matrixpreparation.Chicken ovalbumin, a well known high-mannose, N-linked

glycosylated protein, was used as a quantitative reference. Knownamounts of ovalbumin were spotted on the nitrocellulose mem-brane prior to processing with ['25I]Con A. In comparison to theCon A binding affinity of the known amount of ovalbumin, therelative intensity of ['25I]Con A binding to polypeptides in theglyoxysomal track suggested that the glyoxysomal proteins ac-counted for less than 10% of the protein in the sample (100 ,gwere loaded on each track) (Fig. 3).

Incorporation of Glucosamine and N-Acetylglucosamine. En-dosperm halves from 2-d-old seedlings previously treated with100gM GA3 were exposed to ['4C]glucosamine for 9 h. Labeled

'Abbreviation: Con A, concanavalin A. glyoxysomes were isolated on gradients and matrix polypeptides

951

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Plant Physiol. Vol. 80, 1986

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FIG. 1. Electron micrograph of glyoxysome preparation (x 55,000).

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FIG. 2. Western blot of glyoxysomal matrix polypeptides decoratedby ['25IJCon A. ['25I]Con A binding was carried out in the absence (A)or presence (B) of a-methylglucoside, 30 mg/ml.

processed for acrylamide gels and fluorography. Results showed8 to 10 polypeptides were labeled by glucosamine (Fig. 4).

Hydrolysis of isolated glyoxysomes labeled by [3HJGlcNAcwas carried out to determine the fate of the label. The resultsindicated that the label in the glycoprotein hydrolysate remainedin a discrete component which co-migrated with authentic stand-ard (data not shown).

FIG. 3. Affinity chromatography on lac-agarose. Glyoxysomal poly-peptides were loaded on an acrylamide gel before (lanes A and C) orafter (lanes B and D) elution from lac-agarose. Western blots wereexposed to ['25I]Con A in the absence (lanes A and B) or presence of a-methylglucoside (lanes C and D). Ovalbumin was spotted directly on thenitrocellulose prior to treatment with ['25I]Con A. '4C-mol wt markerswere run in parallel tracks but not subjected to ['25I]Con A.

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GLYOXYSOMAL MATRIX GLYCOPROTEINS

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hoursFIG. 5. Incorporation of [3H]glcNAc in endosperm tissue slices. The

tissue was preincubated for h in the presence (50 ug/ml) (0) or absence(0) of tunicamycin. Labeled proteins were immunoprecipitated with anantibody directed against glyoxysomal matrix proteins, washed withphosphate-buffered saline, and counted by liquid scintillation.

14.3-

FIG. 4. Fluorograph of glyoxysomal polypeptides labeled in vivo by['4C]glcN. Two-day old seedlings were pretreated with 100 Mm GA3 for12 h. Endosperm halves were excised and their adaxial surfaces exposedto ['4C]glcN (5 uCi/endosperm halt) for 9 h. Glyoxysomal polypeptideswere precipitated with acetone and loaded on the acrylamide gel. Filmwas exposed for 7 d.

To determine whether N-GlcNAc was incorporated into an N-linked oligosaccharide, labeling was carried out in the presenceor absence of tunicamycin, a well characterized inhibitor ofdolichol-mediated N-linked glycosylation (19). [3H]GlcNAc wasapplied to thin slices of endosperm tissue and sampled at 2-hintervals. Glyoxysomal proteins were precipitated by a polyspe-cific antibody to glyoxysomal matrix proteins. The results dem-onstrated that incorporation of [3H]GlcNAc into glyoxysomalglycoproteins is linear for at least 6 h (Fig. 5). Inhibition of [3H]GlcNAc incorporation by tunicamycin was evident (Fig. 5) underconditions which did not inhibit the incorporation of [3H]leucine(data not shown).Glyoxysomal Polypeptides in ER Vesicles. Since glycosylation

takes place in the ER it was of interest to determine whetherpolypeptides comparable to those in the glyoxysomal matrixwere detectable in ER vesicles. Polypeptides from ER vesiclesand glyoxysomal matrix which had been labeled in vivo by [35S]methionine were immunoprecipitated with antiserum to glyox-ysomal matrix proteins (Fig. 6). Comparison of polypeptides inER vesicles (lane B) with the corresponding glyoxysomal prepa-ration (lanes A and C) demonstrated that ER vesicles containeda large number of polypeptides which crossreacted with theantiglyoxysomal matrix proteins antibody.

DISCUSSIONThe occurrence of glycoproteins in the matrix of glyoxysomes

only minimally contaminated with extraneous organelles hasbeen demonstrated. At least eight glycoproteins ranging in sizefrom 39 to 160 kD were found. Incorporation of N-acetylgluco-samine into. matrix proteins, inhibition of this process by tuni-

camycin, and the affinity of the polypeptides for Con A allindicate that these glycoproteins are N-linked, mannose-contain-ing glyco-conjugates.

Previous efforts to detect glycoproteins in the glyoxysomalmatrix have involved searches for protein-conjugated oligosac-charides (a) on specific, purified enzymes (10, 28, 32, 38), and(b) more generally, among components of glyoxysomal extracts(3, 32, 33). Malate synthase of castor bean was initially thoughtto be glycosylated (32). However, subsequent analyses led to theopposite conclusion (28). Isocitrate lyase from cucurbits has beenanalyzed by various means which have also resulted in conflictingreports of the presence of carbohydrate substituents (1, 10, 38).

Bergner and Tanner (3) examined isolated glyoxysomes afterincorporation of -[ 1- 4C]glucosamine (57 Ci/mol). These work-ers were able to show incorporation of label into the membranefraction but not into the matrix components. While these resultsare at variance with those reported in this paper, differences inexperimental design could provide an explanation for the con-flicting data. For example, the tissue used in these experiments(Fig. 4) was labeled for 9 h with D_[U-14C]glucosamine of highspecific activity (277 Ci/mol). Another important differenceinvolves the procedure for separation of membrane and matrixproteins. Bergner and Tanner diluted glyoxysomes to 12.5 mMTricine buffer (pH 7.5) (cf to 0.2 M KCI, see "Materials andMethods") which they indicated, yielded approximately 50% ofthe organelle protein in the membrane fraction. This suggeststhat a good portion of the matrix and peripheral components(considered as matrix in this paper), including the glycoproteins,in their preparation remained with the membrane.

Localization of glycoproteins in the glyoxysomal matrix pro-vides an explanation for the presence, during early germination,of substantial proportions of glyoxysomal proteins in the ER(15-17, 21). Since the capacity for core glycosylation is confinedto the ER in castor bean endosperm (30, 34-37), it follows thatthe glyoxysomal glycoproteins must be routed through the ER.This point is important in that it provides insight into the pathof the glyoxysomal proteins from site of synthesis to final desti-nation. A complete understanding of the path of protein trans-port is necessary if we are to deduce the signals which directprotein traffic into specific organelles.The model depicted in Figure 7 takes into account several

important facts: (a) that synthesis ofglyoxysomal enzymes takesplace on nonmembrane bound polysomes (39); (b) that glyoxy-somal matrix proteins are glycosylated (this paper); (c) that ERis the site of glycosylation in castor bean endosperm (30, 34);

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GONZALEZ

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Plant Physiol. Vol. 80, 1986

A Focalized appearance ofER membrane receptors J

A A AE

ER i

8Cytosolic synthesis of lglyoxysome matrix enzymes

ICPost-tronslationalsequestration andglycosylation of glyoxysomematrix proteins in ER

transit peptide?

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Glyoxysome

_ eAAla ~ lcp

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L matrix of mature glyoxysome

FIG. 7. Schematic representation of glyoxysomal biogenesis.

Since a single N-linked oligosaccharide can increase the mass ofa protein by approximately the equivalent of 20 amino acids,the possibility of proteolytic or glycosidic processing of glyoxy-somal glycoproteins seems worthy of direct investigation.

Acknowledgment-I thank M. D. Brush for excellent technical assistance.

FIG. 6. Fluorograph of labeled ER and glyoxysomal polypeptides inan SDS-polyacrylamide gel. The proteins were labeled in vivo with [3'S]methionine and immunoprecipitated from isolated glyoxysomes (lanesA and C) and isolated ER (lane B) using an antibody directed againstglyoxysomal matrix proteins. The positions of glycoprotein bands iden-tified by ['25I]Con A (Fig. 2) are indicated by open arrows. Closed arrowsidentify radioactive polypeptides present in the glyoxysomal extracts butnot in ER vesicles.

and (d) that the ER is the origin of the glyoxysomal membrane(5, 8, 12, 27; E Gonzalez, unpublished data). The model doesnot exclude the possibility that some nonglycosylated compo-nents might be taken directly from the cytosol into matureglyoxysomes nor does it eliminate the possibility that transit-likesequences might be necessary for recognition by appropriatereceptors. However, the model does predict the existence ofappropriate ER-associated receptors for glyoxysomal polypep-tides. Receptors with either homogenous or heterogenous distri-bution could be hypothesized among elements ofthe ER. Recentstudies indicate that the ER is heterogenous with respect tophospholipid-synthesizing enzymes (7) thus suggesting the pos-sibility of structural microdomains within the ER network.A transit peptide has been described for glyoxysomal malate

dehydrogenase (1 1). According to recent studies, this sequenceappears to be important in effecting proper translocation of theenzyme across the glyoxysomal membrane (18). If confirmed,this finding would indicate that the transit peptidase is intrinsicto the glyoxysomal membrane. It would be of great interest todetermine whether recognition of the transit peptide depends onconserved sequences also present on other glyoxysomal enzymes.

LITERATURE CITED

1. BECKER WM, H RIEZMAN, EM WEIR, DE TITUS, CJ LEAVER 1982 In vitrosynthesis and compartmentalization of glyoxysomal enzymes from cucum-ber. Ann NY Acad Sci 386: 329-348

2. BEEVERS H 1982 Glyoxysomes in higher plants. Ann NY Acad Sci 386: 243-251

3. BERGNER U, W TANNER 1981 Occurrence of several glycoproteins in glyoxy-somal membranes of castor beans. FEBS Lett 131: 68-72

4. BONNER WM, RA LASKEY 1974 A film detection method for tritium labeledproteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46: 83-88

5. BOWDEN L, JM LORD 1976 Similarities in the polypeptide composition ofglyoxysomal and endoplasmic reticulum membranes from castor bean en-dosperm. Biochem J 154: 491-499

6. BURRIDGE K 1978 Direct identification of specific glycoproteins and antigensin sodium dodecyl sulfate gels. Methods Enzymol 50: 54-64

7. CONDER MJ, JM LORD 1983 Heterogenous distribution ofglycosyltransferasesin the endoplasmic reticulum of castor bean endosperm. Plant Physiol 72:547-552

8. DONALDSON RP, H BEEVERS 1977 Lipid composition of organelles fromgerminating castor bean endosperm. Plant Physiol 59: 259-263

9. DONALDsoN RP, H BEEVERS 1978 Organelle membranes from germinatingcastor bean endosperm. I. A comparison of purified endoplasmic reticulum,glyoxysomes and mitochondria. Protoplasma 97: 317-327

10. FREVERT J, H KINDL 1978 Plant microbody proteins. Purification and glyco-protein nature of glyoxysomal isocitrate lyase from cucumber cotyledons.Eur J Biochem 92: 35-43

1 1. GIETL C, B HOCK 1982 Organelle-bound malate dehydrogenase isozymes aresynthesized as higher molecular weight precursors. Plant Physiol 70: 483-487

12. GOLDBERG DB, E GONZALEZ 1982 A comparison of castor bean endoplasmicreticulum and glyoxysome intrinsic membrane proteins. Ann NY Acad Sci386: 502-503

13. GONtALEZ E 1978 Effect of gibberellin A3 on the endoplasmic reticulum andon the formation of glyoxysomes in the endosperm of germinating castorbean. Plant Physiol 62: 359-362

14. GONZALEZ E 1981 Glyoxysome biogenesis in castor bean. Plant Physiol 67: S.47

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GLYOXYSOMAL MATRIX GLYCOPROTEINS

15. GONZALEZ E 1982 Aggregated forms ofmalate and citrate synthase are localizedin the endoplasmic reticulum of endosperm of germinating castor bean.Plant Physiol 69: 83-87

16. GONZALEZ E 1982 Localization of malate synthase in the cisternae of endo-plasmic reticulum in endosperm of castor bean. Ann NY Acad Sci 386:488-490

17. GONZALEZ E, H BEEVERS 1976 Role of the endoplasmic reticulum in glyoxy-some formation in castor bean endosperm. Plant Physiol 57: 406-409

18. HOCK B 1984 Processing and organelle import of malate dehydrogenaseisoenzymes: Is there a common precursor for the glyoxysomal and mito-chondrial forms? Physiol Veg 22: 333-339

19. HORI H, AD ELBEIN 1981 Tunicamycin inhibits protein glycosylation insuspension cultured soybean cells. Plant Physiol 67: 882-886

20. HUANG AHC, H BEEVERS 1973 Localization of enzymes within microbodies.J Cell Biol 58: 379-389

21. KAGAWA T, E GONZALEZ 1981 Organelle-specific isozymes of citrate synthasein castor bean endosperm. Plant Physiol 68: 845-850

22. KINDL H 1982 Glyoxysome biogenesis via cytosolic pools in cucumber. AnnNY Acad Sci 386: 314-328

23. KINDL H, W KOLLER, J FREVERT 1980 Cytosolic precursor pools duringglyoxysome biosynthesis. Z Physiol Chem 361: 465-467

24. KOLLER W, H KINDL 1978 The appearance of several malate synthase-containing cell structures during the stage ofglyoxysome biosynthesis. FEBSLett 88: 83-86

25. LAEMMLI UK 1970 Cleavage of structural proteins during assembly ofthe headof bacteriophage T4. Nature 277: 680-685

26. LORD JM, L BOWDEN 1978 Evidence that glyoxysomal malate synthase issegregated by ER in castor bean endosperm. Plant Physiol 61: 266-270

27. LORD JM, T KAGAWA, TS MOORE, H BEEVERS 1973 Endoplasmic reticulumas the site of lecithin formation in castor bean endosperm. J Cell Biol 57:659-667

28. LORD JM, LM ROBERTS 1982 Glyoxysome biogenesis via the endoplasmicreticulum in castor bean? Ann NY Acad Sci 386: 362-373

29. MARKWELL MAK 1982 A new solid-state reagent to iodinate proteins. I.

Conditions for the efficient labeling of antiserum. Anal Biochem 125:427-432

30. MARRIoTr K, W TANNER 1979 Dolichylphosphate-dependent glycosyl transferreactions in the endoplasmic reticulum of castor bean endosperms. PlantPhysiol 64: 445-449

31. MARTIN C, JR BEECHING, DH NORTHCOTE 1984 Changes in levels oftranscriptsin endosperm of castor beans treated with exogenous gibberellic acid. Planta162: 68-76

32. MELLOR RB, L BOWDEN, JM LORD 1978 Glycoproteins of the glyoxysomalmatrix. FEBS Lett 90:275-278

33. MELLOR RB, T KRusIus, JM LORD 1980 Analysis ofglycoconjugate saccharidesin organelles isolated from castor bean endosperm. Plant Physiol 65: 1073-1075

34. MELLOR RB, JM LORD 1979 Subcellular localization of mannosyl transferaseand glycoprotein biosynthesis in castor bean endosperm. Planta 146: 147-153

35. MELLOR RB, JM LORD 1979 Involvement ofa lipid-linked intermediate in thetransfer of galactose from UDP-galactose-'4C to exogenous protein in castorbean endosperm homogenates. Planta 147: 89-96

36. MELLOR RB, LM ROBERTS, JM LORD 1979 Glycosylation ofexogenous proteinby endoplasmic reticulum membranes from castor bean (Ricinus communis)endosperm. Biochem J 182: 629-631

37. MELLOR, RB, LM ROBERTS, JM LORD 1980 N-Acetyl glucosamine transferreactions and glycoprotein biosynthesis in castor bean endosperm. J Exp Bot31: 993-1003

38. RIEZMAN H, EM WIER, CJ LEAVER, DE Trrus, WM BECKER 1980 Regulationof glyoxysomal enzymes during germination of cucumber. 3. In vitro trans-lation and characterization of four glyoxysomal enzymes. Plant Physiol 65:40-46

39. ROBERTS LM, JM LORD 1981 Synthesis and posttranslational segregation ofglyoxysomal isocitrate lyase from castor bean endosperm. Eur J Biochem119: 43-49

40. TRELEASE RN 1984 Biogenesis of glyoxysomes. Annu Rev Plant Physiol 35:321-347

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