Evidence for a UDP-Glucose Transporter in Colgi Apparatus ... · Golgi apparatus necessary to overcome this topological problem. Evidence that other proteins distinct from glyco-

Plant Physiol. (1 996) 11 2: 1585-1 594

Evidence for a UDP-Glucose Transporter in Colgi Apparatus-Derived Vesicles from Pea and I t s Possible

Role in Polysaccharide Biosynthesis'

Patricia MUAOZ, Lorena Norambuena, and Ariel Orellana*

Department of Biology, Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile

The Colgi apparatus in plant cells is involved in hemicellulose and pectin biosynthesis. While it is known that glucan synthase I is responsible for the formation of P-1-4-linked glucose (Clc) polymers and uses UDP-Clc as a substrate, very little is known about the topography of reactions leading to the biosynthesis of polysaccharides in this organelle. W e isolated from pea (Pisum sativum) stems a fraction highly enriched in Colgi apparatus-derived vesicles that are sealed and have the same topographical orientation that the membranes have in vivo. Using these vesicles and UDP-Clc, we reconstituted polysaccharide biosynthesis in vitro and found evi- dente for a luminal location of the active site of glucan synthase 1. In addition, we identified a UDP-Clc transport activity, which is likely to be involved in supplying substrate for glucan synthase 1. W e found that UDP-Clc transport is protein mediated. Moreover, our results suggest that UDP-Clc transport is coupled to the exit of a luminal uridine-containing nucleotide via an antiporter mechanism. W e suggest that UDP-Clc is transported into the lumen of Colgi and that Clc is transferred to a polysaccharide chain, whereas the nucleotide moiety leaves the vesicle by an antiporter mechanism.

The Golgi apparatus of growing plant cells is mainly involved in the biosynthesis of hemicellulose and pectin, both components of the primary cell wall (Driouch et al., 1993; Gibeaut and Carpita, 1994). Most of the analyses regarding the biosynthesis of hemicellulose have been done in Golgi membrane fractions from pea (Pisum sativum; Ray et al., 1969; Brummell et al., 1990; White et al., 1993) and soybean (Glycine max; Hayashi and Matsuda, 1981). These studies have shown that GS-I, a /3-1-4-glucosyltransferase probably involved in xyloglucan biosynthesis, is associated with the Golgi apparatus. To our knowledge, this enzyme has not been purified and nothing is known about its orientation in the Golgi apparatus, although it is likely that glycosyltransferases work in the lumen of the organelle, since the product of these enzymes, hemicellulose, has been found in the lumen of Golgi cisternae by immunoelectron microscopy (Zhang and Staehelin, 1992).

UDP-Glc, which is the substrate for GS-I, is located in the cytosol of the plant cell (Hayashi, 1989). However, the putative product of GS-I is found in the lumen of the Golgi

This work was supported by grant Fondecyt 1940571 Chile, Fundación Andes, Chile, and PEW Latin American Fellows Program.

* Corresponding author; e-mail [email protected]; fax 56-2-2712983.

apparatus (Zhang and Staehelin, 1992). This poses a topological problem and it is possible to postulate that polysaccharide biosynthesis requires, in addition to the glycosyltransferases, other factor(s) or protein(s) located in the Golgi apparatus necessary to overcome this topological problem. Evidence that other proteins distinct from glycosyltransferases may be involved in polysaccharide biosynthesis has been presented by Dhugga et al. (1991), who showed that a 40-kD Golgi protein seemed to be involved in polysaccharide biosynthesis. This protein did not correspond to GS-I and it was transiently glucosylated when UDP-Glc was added to Golgi membranes.

In both mammals and yeast, the topography of reactions in the Golgi apparatus leading to glycosylation and mannosylation of proteins has been well characterized (Hirsch- berg and Snider, 1987; Abeijon et al., 1989). They concluded that a11 glycosylation reactions occur in the lumen of Golgi apparatus. In addition, a necessary step for glycosylation and mannosylation reactions is the transport of nucleotide sugars from the cytoplasm into the lumen by specific transporters located in the Golgi membrane. These nucleotide sugar transporters seem to work as antiporters with their respective nucleoside monophosphates (Capasso and Hirschberg, 1984), which are derived from the action of the NDPase located in the lumen of the Golgi apparatus (Brandan and Fleischer, 1982; Abeijon et al., 1989). This enzyme uses as substrate the nucleoside diphosphate gen- erated upon sugar transfer from the nucleotide sugar, and in yeast it has been shown that the Golgi NDPase activity plays an important role in the glycosylation mechanism (Abeijon et al., 1993; Berninsone et al., 1994).

To investigate the mechanism leading to polysaccharide biosynthesis in the plant Golgi apparatus and to detect activities distinct from GS-I that might play an important role in polysaccharide biosynthesis, we reconstituted this process in vitro. We prepared sealed Golgi vesicles and investigated the topography of the reactions leading to the incorporation of Glc, from UDP-Glc, into polysaccharides. Our results suggest that biosynthesis of Glc-containing polysaccharides occurs in the lumen of Golgi apparatus. In addition, we have detected a UDP-Glc transport activity that may be required for substrate availability to the glucosyltransferase-active site. This nucleotide sugar trans-

Abbreviations: GS, glucan synthase; NDPase, nucleoside diphosphate; STM buffer, Suc-Tris-magnesium buffer.

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1586 MuAoz et al. Plant Physiol. Vol. 11 2, 1996

porter, like its counterparts in mammals and yeast, seems to work as an antiporter, since the addition of UDP-Glc stimulates the outward transport of uridine-containing nucleotides from the vesicles.

MATERIALS AND METHODS

UDP-[3H]Glc (4.5 Ci/mmol) was purchased from Am- ersham. UDP-[14C]Glc (300 mCi / mmol) and [14C]Glc-1-P (300 mCi/ mmol) were from American Radiolabeled Chemicals (St. Louis, MO). [I4C]G1c (242 mCi/mmol) was purchased from ICN. Nonradioactive UDP-Glc, cellulase from Trichoderma reseii, pronase E, and buffers were purchased from Sigma. SUC was of high purity and purchased from ICN.

Synthesis of [3H]UDP-Glc

[3H]UDP-Glc was synthesized as described by Dhugga and Ray (1994). Briefly, 250 pCi of [3H]UTP (New England Nuclear; 37 Ci/mmol) was dried under nitrogen gas, resuspended in 200 pL of 40 mM Mops, pH 7.2, and 5 mM MgCl,, 15 mM Glc-1-P, 5 units of UDP-Glc pyrophospho- rylase (Sigma), and 5 units of inorganic pyrophosphatase (Sigma) were added. The mixture was incubated at 35°C for 30 min, 5 mg of activated charcoal was added, the sample was centrifuged, and the pellet was washed twice with 1 mL of ice-cold 1 mM Tris-HC1, pH 7.5. [3H]UDP-Glc was extracted by 200-/.~L extractions with 50% (v/v) etha- no1 containing 0.16 M NH40H. The first and second extractions were pooled and used. The identity of [3H]UDP-Glc was confirmed by comparing the migration of radioactivity with standards in two different TLC systems: polyethylene imide-cellulose plates (Aldrich) using 0.3 M LiCl as solvent and polyethylene imide-cellulose using 1 N acetic acid for 2 cm and then 1 N acetic acid-LiC13 M (90:1O, v/v) for 15 cm. In both systems the radioactivity co-migrated with the UDP-Glc standard. In addition, acid and alkali treatment of the sample, as described by Paladini and Leloir (1952), produced UDP and UMP, respectively, as expected for UDP-Glc.

Golgi-Vesicle Preparation

The method to obtain Golgi-derived vesicles was based on the Leelavathi procedure (Leelavathi et al., 197O), with minor modifications. Pea seedlings (Pisam sativum var Alaska) were grown in moist vermiculite for 7 d in the dark at 25°C. Stem segments (1 cm) were excised from the elongating region of the hypocotyls. The tissue was kept on ice until homogenization. In different preparations 40 to 70 g of tissue was homogenized by hand with razor blades in the presence of 1 volume of 0.5 M SUC, 0.1 M KH,P04, pH 6.65, 5 mM MgCl,, and 1 mM DTT at 4°C. When the tissue was finely chopped, it was homogenized for 3 min in a mortar. This procedure was done on ice. The homogenate was filtered through Miracloth (Calbiochem) and centrifuged at 1,OOOg for 5 min in a rotor (JA-20, Beckman). The supernatant was loaded on an 8-mL 1.3 M SUC cushion and

centrifuged at 100,OOOg in a rotor (AH-629, Sorvall) for 70 min. The upper phase was removed without disturbing the interface fraction. Additionally, a discontinuous gradient was formed by adding 1.1 and 0.25 M SUC and centrifuged for 90 min at 100,OOOg in the AH-629 rotor. The interface at 0.2511.1 M SUC fraction was collected, diluted in 1 volume of distilled water, and centrifuged at 100,OOOg in a rotor (T647.5 Sorvall) for 35 min. The pellet was resuspended using a Dounce homogenizer in a buffer containing 0.25 M

SUC, 1 mM MgCl,, and 10 mM Tris-HC1, pH 7.5. The vesicles were kept at -70°C until use.

linear SUC Gradients

SUC gradients (20-50%, w / w) were prepared according to the method described by Hayashi et al. (1988). Pea stems were homogenized as above in a buffer containing 50 mM Hepes/KOH, pH 7.0, 0.4 M SUC, 10 mM KC1, 1 mM MgCl,, 1 mM EDTA, and 1 mM DTT. The gradient (25 mL) was made in a buffer containing 50 mM Hepes/KOH, pH 7.0, 0.1 mM MgCl,, 1 mM EDTA, and 1 mM DTT. Ten milliliters from the 1,OOOg supernatant described above was loaded on top and centrifuged for 3.5 h at 100,OOOg in the AH-629 rotor. Fractions of 1.5 mL were collected.

Enzyme and Protein Assays

UDPase in the presence and absence of 0.1% (v/v) Triton X-100, Cyt c oxidase, and NADH Cyt c reductase insensitive to antimycin A were measured as described by Briskin et al. (1987). GS-I and GS-I1 were measured as described by White et al. (1993), except that no carrier was added. Pro- teins were measured by the bicinchoninic acid method (Pierce).

lncorporation of UDP-Clc into Vesicles

One hundred micrograms of Golgi-vesicle protein was incubated in a final volume of 100 pL, with 1 /.LM of UDP-[3H]Glc (1 X 106 cpm/mL) in a buffer containing 0.25 M SUC, 1 mM MgCl,, and 10 mM Tris-HC1, pH 7.5 (STM buffer). Incubation was for 3 min (or as indicated else- where) at 25OC. To stop the incubation the sample was diluted with 10 volumes of ice-cold STM buffer and filtered through 0.7-pm glass fiber filters or 0.45-pm nitrocellulose filters in a filtration system (Hoefer, San Francisco, CA). Subsequently, the filters were washed with an additional 10 volumes of ice-cold STM buffer, dried, and counted with scintillation liquid. The separation by filtration of the vesicles from the incubation medium took no longer than 20 s.

Pronase E Treatment of the Vesicles

One milligram of Golgi-vesicle protein was incubated in the absence or presence of 10 or 50 pg of pronase E (Sigma) at 30°C in a final volume of 500 pL. After 10 min, 4 volumes of ice-cold STM buffer was added and centrifuged at 100,OOOg for 30 min at 4°C. The pellet was surface-washed and resuspended in 250 pL of STM buffer and immediately used for the different assays.



Topography of Polysaccharide Biosynthesis in Plant Colgi 1587

Cellulase Digestion

One hundred micrograms of Golgi-vesicle protein was incubated at 25°C for 3 min in a final volume of 100 pL in STM buffer containing 1 p~ UDP-[3H]Glc. The incubation was stopped with 10 volumes of ice-cold STM buffer and then boiled for 1 min. The pH was adjusted to pH 5.0 by addition of 100 mM sodium acetate. Next, 3.8 units of cellulase from Trichoderma reseii (Sigma) was added and incubated for 18 h under toluol. Ethanol was added to a 70% (v/v) final concentration, boiled for 1 min, chilled for 15 min on ice, and then filtered through 1.5-pm glass fiber filters. The filters were washed with 4 mL of ice-cold 70% (v/v) ethanol and dried, and the radioactivity was determined by liquid scintillation spectroscopy. Control samples were done as described above but no cellulase was added or boiled Golgi vesicles were used in the 3-min incubation.

A11 the experiments were done at least twice, and every test was performed with duplicate or triplicate assays.

RESULTS

Preparation of Golgi-Derived Vesicles

To investigate the topography of reactions involved in polysaccharide biosynthesis in the Golgi apparatus, it was necessary to obtain a highly enriched, sealed Golgi-vesicle fraction with the same topology as in vivo. To obtain these vesicles, we found that a modification of the Leelavathi procedure (Leelavathi et al., 1970) was the best in terms of yield, topology, and vesicle latency (Table I). The characterization of this fraction gave an enrichment of 60- to 90-fold over homogenate in GS-I, a known Golgi marker enzyme. We analyzed the yield in terms of protein from different preparations, and we found that by starting with 40 to 70 g of stems we obtained 1 to 2 g of protein in the homogenate and 5 to 15 mg of total protein in the Golgi- vesicle fraction. Contamination by other organelles was detected by marker enzyme activities. Taken as 100% of the activity present in the homogenate, 10 to 15% of antimycin A-insensitive NADH-Cyt c reductase (ER marker enzyme), 1% Cyt oxidase (mitochondrial marker enzyme), and 5% GS-I1 (plasma membrane marker enzyme) were present in the Golgi-vesicle-enriched fraction. To establish the orientation and integrity of the Golgi vesicles, we measured an

Table 1. Preparation of a Golgi-enriched-derived vesicle fraction from pea stem

Results from a representative Golgi preparation are shown.

Determination Homorenate Golri-Enriched Fraction

Protein (mg) N1257 5.8

GS-I (total pmol) 404 166 GS-l (pmol mg-' min-') 0.01 6 1.445

UDPase-Triton X - I O0 (nmol) - 37 UDPase + Triton X-100 (nmol) - 182 Latency - 80%

a -, Not determined.

200

s

" I

O 5 I 0 1 5 Time (min)

Figure 1. lncorporation of UDP-[3H]Glc into Golgi vesicles and ethanol-insoluble products versus time. Golgi vesicles (1 O0 pg) from pea stems were incubated at different times in a medium containing 1 p~ UDP-Glc. lncorporation of UDP-[3H]Gk into Golgi vesicles (I, specific activity 874 cpm/pmol) and incorporation of UDP- [3HlGlc into 70% (v/v) ethanol-insoluble products (A, specific activity 1040 cpm/pmol) were measured using filtration. Results are means t SE.

enzyme with an active site that faces the lumen of the vesicles. In this case, sealed, right-side-in vesicles should not exhibit activity, and activity would be evident only upon disruption of the membrane. An e n z y m e that fit these characteristics is latent UDPase (or latent IDPase), which is described as a marker enzyme for plant-derived Golgi (Ray et al., 1969), and it seems to be active in the lumen of Golgi cisternae (Dauwalder et al., 1969). The latency of severa1 vesicle preparations was between 80 and 90% (Table I), which demonstrates that 80 to 90% of the vesicles were sealed and had the same orientation. Thus, we obtained an enriched Golgi-vesicle preparation suitable to perform topographical analysis.

lncorporation of UDP-Glc into Vesicles and Polysaccharide Synthesis

When vesicles were incubated with UDP-[3H]Glc and quickly separated from the incubation medium by filtration, radioactive label from UDP-Glc was incorporated into Golgi vesicles in a time-dependent manner (Fig. 1, squares). It is well known that UDP-Glc is utilized by the Golgi apparatus from pea stems for polysaccharide biosynthesis; therefore, it was important to determine whether transfer of Glc into polysaccharides occurs in this in vitro system under the assay conditions. We incubated Golgi vesicles with UDP-[3H]Glc at various times, but instead of filtering the vesicles, we stopped the reactions by adding 70% (v/v) ethanol, followed by filtration. Under these conditions any 3H radioactivity associated with the filters is likely to be polysaccharide-bound (White et al., 1993). We found that more than 70% of the label incorporated into Golgi vesicles was ethanol-insoluble (Fig. 1, triangles). To analyze whether this ethanol-insoluble product was indeed polysaccharides, we treated the [3H]Glc-containing mate-



1588 MuRoz et al. Plant Physiol. Vol. 11 2, 1996

ria1 formed in the Golgi vesicles with cellulase from T. reseii and found that most of the material was sensitive to this enzyme (Fig. 2). These results strongly suggest that most of the ["HIGlc from UDP-["H]Glc is transferred to polysaccharides in the vesicles. Given the cellulase sensitivity and the incubation conditions, i.e. 1 PM UDP-Glc and 1 mM MgCl,, it is likely that vesicles form P-1-4-linked polymers of Glc due to GS-I activity located in the Golgi apparatus of pea stems (Ray et al., 1969). This Glc-containing polysaccharide resembles the backbone structure of xyloglucan, a hemicellulose that is known to be synthesized in the Golgi apparatus of pea stems (Brummell et al., 1990; White et al., 1993). However, we cannot rule out that another type of Glc polymer, which is sensitive to the commercial preparation of cellulase from T. reseii, is also formed by the vesicles. We looked for incorporation of Glc into lipids as described by Hayashi and MacLachlan (1984), but we were unable to detect it (data not shown).

The above results confirm that Golgi vesicles utilize UDP-Glc to transfer Glc into polysaccharides. Since Glc- containing polysaccharides are located in the lumen of the Golgi apparatus (Zhang and Staehelin, 1992), one possibility is that polymerization takes place in the lumen of the Golgi vesicles, but the substrate for polysaccharide biosynthesis is outside the vesicles. Therefore, in addition to polymerization, UDP-Glc transport could be required for polysaccharide biosynthesis in Golgi vesicles. What this means is that the UDP-Glc incorporation assay into Golgi vesicles using UDP-[3H]Glc probably measures a combina- tion of different processes or steps.

We observed the following characteristics for the incorporation of UDP-[3H]Glc into Golgi vesicles and polysaccharides. T h e total process was sensitive to t empera tu re (Fig. 3A), since incubation at 0°C as well as pretreatment of the vesicles for 5 min at 100°C abolished UDP-Glc incorporation. Permeabilization of the vesicles with detergent

Figure 2. Radioactivity incorporated into pea stem Golgi vesicles is sensitive to cellulase. Pea stem Colgi vesicles (100 pg) were incubated with UDP-13H]Glc (specific activity 11 57 cpm/pmol) for 3 min, and the reaction was stopped by boiling. Afterward, the sample was incubated in the absence or presence of cellulase from T. reseii and the product insoluble in 70% ethanol was determined by filtration. The control was done using boiled Golgi vesicles in the incubation medium. Results are means 2 SE.

60

E -0 3

3

.- E 5 40 B

2. s F

20

O

Figure 3. lncorporation of UDP-Glc and putative UDP-Glc breakdown products into Colgi vesicles. A, Effect of temperature and detergent. Colgi vesicles (100 pg) were incubated with 1 p~ UDP- [3HIGlc (specific activity 492 cpm/pmol) for 3 min under different conditions: 25"C, normal conditions; Triton X-1 00, incubation in the presence of 0.1 O/O (v/v) Triton X- l 00; O"C, incubation in an ice bath; and 100°C, vesicles boiled for 5 min prior to the assay. B, Colgi vesicles (100 pg) were incubated separately for 3 min under the standard conditions in the presence of UDP-['4C]Clc (specific activity 181 5 cpm/pmol), ['4ClGlc (specific activity 3575 cpm/pmol), or ['4C]Glc-1 -P (1 655 cpm/pmol) at 1 p~ final concentration. Incorpo- ration into Colgi vesicles was measured by a filtration assay. Results are means t SE.

had no effect on UDP-[3H]Glc incorporation into vesicles (Fig. 3A). The experiments in which permeabilized vesicles were used were repeated using ultracentrifugation to sep- arate the vesicles and we obtained similar results to the experiments in which the filtration assay was used (not shown). These results indicate that in permeabilized Golgi vesicles ["HIGlc is also transferred from UDP-[3H]Glc into endogenous polysaccharides, which are re ta ined in the permeabilized vesicles.

To address the possibility that the incorporation of 3H-radioactive material into vesicles and polysaccharides was not due to UDP-Glc itself but to incorporation of potential breakdown products of UDP-Glc, we assayed incorporation of [14C]G1c and ['4C]Glc-1-P into vesicles (Fig. 3B). Very low incorporation was detected compared with the incorporation using UDP-[ 14C]Glc. This result demonstrates that UDP-Glc is utilized by the Golgi vesicles for polysaccharide biosynthesis and not its breakdown products.

Specificity of UDP-Glc lncorporation into Colgi Vesicles

The Golgi-vesicle preparation is highly enriched in Golgi vesicles, but there is some contamination with other organelles. To address the question of whether the incorporation of UDP-Glc into Golgi vesicles was indeed a Golgi- specific process, we fractionated the organelles from pea stem cells on linear Suc gradients and analyzed the incorporation of UDP-["H]Glc in the different fractions of the gradient (Fig. 4). We found a major peak of UDP-Glc incorporation at approximately 33% Suc (1.14 g cm-"). In the same region of the gradient we also detected latent UDPase and GS-I activities, both Golgi markers (Briskin et al., 1987). This result confirms that UDP-Glc incorporation takes place in the Golgi apparatus. In addition to latent



Topography of Polysaccharide Biosynthesis in Plant Golgi 1589

b 2 1.0

Y - m C # 0.5

u. E 0.0

E 0.9 C O Ic

o C m

0.6

g v) n a 0.0

UDP-Glc Inrmo. A

5 10 15 20

Fractions

Figure 4. Subcellular fractionation of pea stems on linear SUC gradients. A, UDP-[3HIClc incorporation into vesicles (m), Cyt c oxidase (MT, mitochondria; A), NADH Cyt c reductase insensitive to antimycin A (ER, O) B, UDPase activity measured in the presence (m) and absence ( O ) of 0.1 O/O Triton X-1 00. C, GS-I ( A ) . D, SUC concentration (O) determined by refractometry.

0 6.0 = -L

-2.5 ??

.o

O

F 8

-25 $ a? (P

kl 5 10 15

UDPase, we detected another UDPase activity in a less dense SUC fraction (25% SUC, 1.10 g ~ m - ~ ) . This UDPase activity is different from the Golgi UDPase in that it is not latent and has a different substrate specificity compared with Golgi UDPase (A. Orellana, P. Muiíoz, and L. Noram- buena, unpublished data). A similar finding regarding other UDPase activities of pea stem was reported by White et al. (1993).

In addition to the co-localization with Golgi markers, we found that there was little or no incorporation of UDP-Glc associated with mitochondria. A minor peak of activity was observed in the region where the ER marker enzyme is detected. However, it is likely that some activity should be located in ER membranes because glycosylation of proteins with Glc-containing glycans takes place in the ER. Since the Golgi-containing fractions from the linear SUC gradient are the main sites of incorporation of UDP-Glc and since our vesicle preparation was highly enriched in Golgi vesicles and contained only 10 to 15% of the total amount of ER, we are confident that the UDP-Glc incorporation in the enriched Golgi-vesicle fraction is a Golgi-specific process.

Topography of GS-I

Where in a Golgi vesicle does the transfer of Glc from UDP-Glc to polysaccharides occur? To investigate the topography of the active site of GS-I and to study whether Golgi membrane proteins facing the cytoplasm could be

20

involved in the reactions leading to polysaccharide biosynthesis, Golgi vesicles were treated with the protease pronase E under conditions that did not damage the integrity of the vesicles. The vesicles were then re-isolated and GS-I activity and the incorporation of UDP-[3H]Glc into vesicles were determined. To ensure that the pronase E treatment did not break the vesicles, we first measured UDPase latency following pronase E treatment. The incubation and re-isolation of the vesicles resulted in a decrease of 85 to 70% of UDPase latency; however, this decrease was the same in control and in pronase E-treated vesicles (Table 11).

Protease treatment decreased GS-I activity to 22% of the control values, which was dependent on protease concentration. When UDP-Glc incorporation was measured, we found a decrease of incorporation that was similar to that of GS-I activity. These results indicate that UDP-Glc incorporation and GS-I activity are related and suggest that a membrane protein facing the cytoplasm is required for normal function of GS-I. Two possibilities could account for these results. Either GS-I is directly affected by pronase E or the target of protease action is a membrane protein, such as a UDP-Glc transporter, involved in the access of UDP-Glc to the lumen, where the active site of GS-I may be located. To differentiate between these two alternatives, we bypassed the putative transport step by permeabilizing the pronase E-treated vesicles with detergent. Upon permeabilization we found that GS-I activity was restored, indicat-

Table II. Effect o f pronase E on incorporation o f UDP-Glc and CS-I activity

One milligram of pea stem Golgi vesicles was incubated in the absence (-) or presence (+) of pronase E, and activity of latent UDPase, UDP-Clc incorporation (UDP-[3HlGlc specific activity 895 cpm/pmol), and CS-I (UDP-[3HlGlc specific activity 1283 cpm/pmol) were determined. The number in parentheses is the percentage of activity, using 100% as the activity of the intact vesicles and without pronase treatment. Tx-1 00. Triton X-100.

UDP-Glc lncorporation GS-l Treatment UDPase Latency

-Tx-1 O 0 +Tx-1 O0 -Tx-1 O 0 +Tx-l O0

% pmol mg-’ 3 min-’ pmol-’ mg-’ 10 min-’ Control 67.3 77.2 (100) 84.7 (109) 77.6 (100) 72.7 (93.7) 10 pg Pronase 67.6 19.2 (24.8) 50.6 (65.5) 17.1 (22.0) 40.8 (52.6) 50 pg Pronase 70.0 32.0 (41.4) 55.1 (71.4) 29.8 (38.4) 49.6 (63.9)



1590 MuRoz et al. Plant Physiol. Vol. 11 2, 1996

ing that the active site was protected from proteolysis (Table 11). However, restoration of GS-I activity was not complete, which could be due to damage of luminal GS-I caused by proteolysis, since the latency of the pronase E-treated vesicles was only 70%, and broken or inside-out vesicles would suffer degradation of luminal proteins to some extent. This is supported by the fact that upon permeabilization both the GS-I activity and UDP-Glc incorporation were restored to 60 to 68% of the control. This value is close or similar to UDPase latency of the pronase E-treated vesicles, suggesting that about 30% of the activity was inactivated by proteolysis. However, we cannot rule out that protease treatment affects a cytosolic portion of the GS-I protein or another protein that could be important for GS-I activity. UDP-[3H]Glc incorporation into permeabilized pronase E-treated vesicles was also restored, in a fashion similar to the restoration seen for GS-I activity (Table 11). This result supports the idea that UDP-[3H]Glc added to vesicles is utilized by GS-I and therefore the UDP-[3H]Glc incorporation assay partly reflects the GS-I activity.

The above results lead us to conclude that the active site of GS-I faces the lumen of the Golgi apparatus and that an externally located membrane protein is required for normal GS-I activity. Since permeabilization of vesicles overcomes the decrease in GS-I activity on pronase E-treated vesicles, it is likely that this membrane protein is a UDP-Glc transporter involved in substrate availability for GS-I.

UDP-Clc Transport

Our experiments in which we used UDP-[3H]Glc showed t h a t m o s t of t h e [3H]Glc is t rans fer red into polysacchar ides and accumulates in the vesicles. To investigate whether UDP-Glc was transported intact into Golgi vesicles, we determined the incorporation of UDP-Glc labeled in the nucleotide moiety. We found that [3H]UDP-Glc was incorporated into Golgi vesicles in a time-dependent fashion and reached an equilibrium at 5 min (Fig. 5A), suggesting that UDP-Glc is indeed transported into the lumen of the Golgi vesicles. To demonstrate that this result was not simply due to binding of UDP-Glc to the Golgi membranes we did two different experiments. First, we permeabilized the vesicles with detergent. In this case, a solute should not remain within the vesicles. Second, we performed the transport assays under different osmotic conditions. This treatment should change the internal volume of the vesicles and it should also change the amount of solute inside the vesicles at equilibrium (assuming that the concentration of substrate at equilibrium does not change under the different osmotic conditions). Jermeabilization decreased the incorporation more than 90% compared with the control, and it was close to background values (Table 111). On the other hand, incorporation of [3H]UDP-Glc was sensitive to changes in osmolarity since incubation in 0.5 M SUC, which should decrease the internal volume of the vesicles, produced a decrease of 30% in the incorporation compared with 0.25 M Suc (Table IV). The results discussed so far make it unlikely that incorporation of [3H]UDP-Glc into Golgi vesicles could be explained simply by binding UDP-

Glc to an externa1 site of the Golgi membrane. But, these data, as well as the lack of incorporation of UDP-Glc breakdown products into Golgi vesicles (Fig. 3B), support the hypothesis that UDP-Glc enters the lumen of Golgi vesicles intact. The incorporation of [3H]UDP-Glc into Golgi vesicles was saturable with an apparent K , of 14 FM and a V,,, of 102 pmol mg-' 3 min-' (Fig. 5B).

Nucleotide Turnover

Our results suggest that UDP-[3H]Glc is transported into Golgi vesicles and that most of the [3H]Glc is transferred to polysaccharides. It is likely, therefore, that soluble UDP will be formed rapidly within the vesicles. This is supported by the experiments reported in Table 111, in which

I I I I

O 5 10 15 20

Time min.

B( 75

I T I

I I I I

O 5 10 15 20

UDP-glucose pM Figure 5 . lncorporation of [3H]UDP-Clc into pea stem Golgi vesicles. A, Pea stem Golgi vesicles (300 pg) were incubated with 1 p~ [3H]UDP-Glc (specific activity 1340 cpm/pmol) at various times as described in "Materials and Methods" and incorporation into Golgi vesicles was determined by filtration. B, Pea stem Golgi vesicles were incubated with various concentrations of [3H]UDP-Clc and incorporation into Golgi vesicles was estimated by a filtration assay. Inset, the reciproca1 plot. Results are means 2 SE.



Topography of Polysaccharide Biosynthesis in Plant Colgi 1591

Table 111. lncorporation of [3HlUDP-Clc is sensitive to detergent

Pea stem Colgi vesicles (300 pg) were incubated with 1 ~ L M [3H1UDP-Clc (specific activity 918 cpm/pmol) for 10 min in the presence (+) and absence (control) of 0.1 % (v/v) Triton X-100. After incubation the samples were filtered and the radioactivity associated with the filters was determined.

Experimental Condition ['H] UDP-Glc lncorporation

pmol mg- ' 10 min- ' Control 6.50 2 0.02 +Triton X-1 O0 1.1 O 2 0.08

permeabilization of vesicles with detergent resulted in a lack of accumulation in the vesicles of [3H]uridine-derived nucleotide. Furthermore, since there is a UDPase activity in the lumen of the Golgi vesicles (Fig. 4), it seems likely that UDP will be hydrolyzed to UMP.

Neither [3H]UDP nor [3H]UMP accumulate to the same extent as [3H]Glc within the Golgi vesicles, since the amount of radioactivity from [3H]UDP-Glc found in the vesicles was much lower than the [3H]Glc transferred into polymers at 15 min of incubation (compare Figs. 1 and 5A). This finding suggests a rapid exit of the [3H]uridine- containing nucleotide. To evaluate this turnover, we used a double-label experiment in which Golgi vesicles were incubated with a mixture of UDP-[14C]Glc plus [3H]UDP-Glc. We then compared the ratio of 14C/3H within the vesicles at 1 and 3 min with the 14C/3H ratio in the incubation medium at zero time. We did this experiment in two different ways. First, under initial rate, we started the incubation with the radioactive mixture containing 1 /.LM UDP- Glc and determined the ratio of 14C/3H at 1 and 3 min. In a different experiment, we incubated the vesicles with cold 1 p~ UDP-Glc for 5 min, the time at which the incorporation of [3H]UDP-Glc reaches equilibrium (Fig. 5A), and then added a trace amount of UDP-[14C]Glc plus [3H]UDP- Glc. If the exit of 3H-nucleotide is slow, at short times, we should see in the vesicles the same 14C/3H ratio as in the incubation medium at zero time. However, if the exit of 3H-nucleotide is fast we should see an increase of the 14C/3H ratio. Under the initial rate conditions at 1 min we saw an increase of the 14C / 3H ratio in the vesicles over the medium, and this increase was even larger at 3 min (Table V, Initial Rate). When the 14C/3H ratio was determined under equilibrium conditions we saw that the 14C/3H ratio at 1 and 3 min also increased compared with zero time. The values at 1 and 3 min were similar and comparable with the value observed at 3 min under initial rate conditions (Table V). These experiments, plus our previous data, indicate that upon UDP-Glc entrance into the Golgi vesicles, Glc is transferred and accumulates in the vesicles as polysaccharides, while the nucleotide moiety leaves the vesicle. How- ever, the comparison between the experiments under initial rate conditions and equilibrium suggests that it is necessary to have a certain intravesicular concentration of the nucleotide moiety to reach the high rate of turnover.

We do not believe that the differences in the 14C/3H ratios between the incubation medium and the vesicles is caused by the entrance of UDP-Glc and its breakdown products at different rates, because that would require the

involvement of UDP-Glc breakdown products as substrates for polysaccharide biosynthesis; the latter is not the case since we have shown that neither Glc nor Glc-1-P are substrates for polysaccharide biosynthesis (Fig. 2). Further- more, we detected only a very small amount of breakdown of UDP-Glc with Glc-I-P formation under the incubation conditions (A. Orellana, P. Muííoz, and L. Norambuena, unpublished results). Moreover, since the double-label experiments were done under conditions of active polysaccharide biosynthesis, the changes in the 14C/3H ratio should be due to entrance and metabolism of UDP-Glc within the vesicles followed by exit of the uridine- containing nucleotide from the vesicles. As we will show below, the exit of the nucleotide moiety may be related to UDP-Glc entry into the vesicles.

UDP-Clc Entry into Colgi Vesicles 1s Coupled to Exit of the Nucleotide Moiety of UDP-Clc

To investigate whether a coupled transport process occurs in plant Golgi vesicles, we loaded the vesicles with [3H]UDP-Glc for 5 min. We knew that by this time that Glc had been transferred and a soluble [3H]-uridine-containing nucleotide had been formed and was at equilibrium (Figs. 1 and 5). We then added a pulse of different nucleotides or nucleotide sugars and measured the nucleotide remaining in the vesicles 3 min after the pulse. We found that UDP- Glc induced a decrease in the radioactive label associated with the vesicles, whereas the addition of t he buffer alone produced almost no effect (Table VI). Different uridine- containing nucleotide sugars, such as UDP-Gal and UDP- Xyl, produced an effect similar to that of UDP-Glc, whereas non-uridine-containing nucleotide sugars, such as GDP- Fuc and ADP-Glc, had almost no effect (Table VI). ADP had no effect, whereas UMP and UDP produced an effect similar to that of UDP-Glc. The results suggest that there is a UDP-Glc transporter in the Golgi membrane and that it works as an antiporter.

DlSCUSSlON

We investigated the topography of Glc-containing polysaccharide biosynthesis in a Golgi-derived vesicle preparation from pea stems. The orientation of these vesicles was determined by measuring latent UDPase. Severa1 lines of evidence indicated that latent UDPase is a membrane pro-

Table IV. lncorporation of [3HIUDP-Glc is sensitive to osmotic changes

Pea stem Golgi vesicles (300 pg) were incubated with 1 p~ I3H1UDP-Glc (specific activity 910 cpm/pmol) in a medium containing 0.25 or 0.5 M SUC. After 3 min of incubation the reaction was filtered and the radioactivity associated with the filters was determi ned.

suc [3Hl UDP-Glc lncorporation

pmol mg-' 3 min-'

0.25 M 5.80 2 0.64 0.5 M 4.09 2 0.34



1592 Muhoz et al. Plant Physiol. Vol. 11 2, 1996

Table V. Ratio o f 14C/3H found in Golgi vesicles after incorporation of a mixture UDP-I'4C]Clc + l3H]UDP-Glc

lnitial rate: Vesicles (100 pg) were incubated with 1 p~ UDP-Glc containing a mixture of UDP-['4CIClc + [3HlUDP-Glc. After 1 or 3 min of incubation the reaction was stopped and filtered, and the radioactivity associated with the filters was determined. Equilibrium: Vesicles were preincubated in the presence of 1 p~ cold UDP-Clc for 5 min and then 2 pL of a mixture containing a trace amount of UDP-['4C]Glc + [3H]UDP-Glc was added, and the incubation continued for 1 or 3 min. The numbers in parentheses are the actual 14C cpmPH cpm and they are the average of duplicate measurements. The SD was less than 10%. The cpms under the "Medium" column were obtained from the incubation medium and corresoond to cDm/uL or cmnhmol.

cpm of ''C/cpm of 3H Experimental Condition

Medium 1 min 3 min

lnitial rate Experiment 1, UDP-['4ClClc + [3H]UDP-Glc 0.82 1.51 2.09

Experiment 2, UDP-['4C]Glc + [3H]UDP-Clc 0.97 1.83 N Da

Equilibrium, UDP-['4ClClc + [3HlUDP-Glc 0.92 2.34 2.42

(807/987) (2067/1369) (62 3 5/2982)

(852/877) (2390/1305)

(637/695) (2699/1151) (6530/2698)

a ND, Not determined.

tein with its active site facing the lumen of the Golgi apparatus. Its activity was unmasked by disrupting the vesicles with detergent. Treatment with protease on intact Golgi vesicles that affect polysaccharide biosynthesis did not affect the UDPase activity to a large extent. In addition, we found that protease treatment decreased the Golgi UDPase activity only in permeabilized vesicles (A. Orel- lana, P. Muííoz, and L. Norambuena, unpublished data). Biochemical and electron microscopy analyses indicate that the plant-derived Golgi NDPase is a membrane protein (Dauwalder et al., 1969; Mitsui et al., 1994). The use of cytochemical techniques followed by electron microscopy localization suggest that Golgi IDPase, which is the same protein as Golgi UDPase (Mitsui et al., 1994), would be active within the cisternae of the Golgi apparatus (Dau- walder et al., 1969). Other Golgi NDPases described in rat liver and yeast also have their active sites facing the lumen of Golgi apparatus (Brandan and Fleischer, 1982; Abeijon et al., 1989).

The Golgi vesicles were able to incorporate Glc from UDP-Glc into polysaccharides. This reaction of sugar transfer is catalyzed by a glucosyltransferase, most likely GS-I. We analyzed the orientation of this enzyme in Golgi vesicles and obtained evidence for a luminal location of the active site of GS-I. Nothing is known about the orientation of glycosyltransferases involved in polysaccharide biosynthesis in the Golgi apparatus from plant cells, and a lumi- na1 localization of the active site of GS-I in pea Golgi apparatus is in agreement with the topological arrange- ment of a number of glycosyltransferases from mammalian Golgi apparatus (Natzuka and Lowe, 1994). Therefore, it seems likely that the topography of polysaccharide biosynthesis resembles the topography of reactions leading to posttranslational glycosylation of N-glycoproteins in the Golgi apparatus from mammalian cells (Hirschberg and Snider, 1987).

Transport of UDP-Glc into the Lumen

The orientation of GS-I in the Golgi vesicles predicts that the substrate must pass through the membranes of Golgi

cisternae and that the transport of UDP-Glc would be a necessary step for the incorporation of Glc into polysaccharides. Our results suggest that UDP-Glc is indeed transported into the lumen of the vesicles, and a Golgi membrane protein is involved in this transport. The transport process was saturable and its K , was in the micromolar range, a characteristic also found in nucleotide sugar transporters described in yeast and mammalian systems (Hirschberg and Snider, 1987; Abeijon et al., 1989). Al- though the presence of nucleotide sugar transporters has been postulated (Gibeaut and Carpita, 1994), to our knowledge this is the first evidence for a Golgi UDP-Glc transporter activity in plants. Moreover, no UDP-Glc

Table VI. Exchange of nucleotides and nucleotide sugars with radiolabeled solutes within Golgi vesicles

Pea stem Golgi vesicles were incubated for 5 min in the presence of 1 p~ [3HlUDP-Glc. At that time the Colgi vesicles were chal- lenged with buffer alone or buffer containing one of the nucleotides or nucleotide sugars (at 1 mM) shown in the table. Three minutes after the challenge (actual time 8 min), the radioactivity st i l l associated with the vesicles was measured by filtration. The control corresponds to the incorporation of [3H]UDP-Glc at 5 min. Each value corresponds to the average of duplicate determinations; the SD of the duplicates was less than 5%. The specific activities of [3Hl UDP-Glc in the three experiments were 1647, 11 90, and 1687 cpm/pmol for experiments I, 11, and 111, respectively.

Challenging Substrate Experiment I Experiment I I Experiment 1 1 1

p m o h g p m o h g p m o h g Control 5.9 6.4 5.8 Buffer 5.5 5.8 6.1 UDP-Glc 2.5 3.7 1.9 UDP-Cal 2.6 3.1 - UDP-XyI 2.6 . 2.7 - ADP-GIc - 5.4 - CDP-Fuc - 5.1 -

UDP 1.9 - - UMP 2.3 - -

AMP 7.2 - - a -, Not determined.

a



Topography of Polysaccharide Biosynthesis in Plant Golgi 1593

transporter has been described in the Golgi apparatus from mammalian or yeast cells.

Since biosynthesis of Glc-containing hemicelluloses is an important function of the Golgi apparatus in growing plant cells, the activity of this transporter in the Golgi apparatus could be highly expressed in tissues that produce hemicelluloses at a high rate. Therefore, it is likely that its expres- sion is regulated in elongating tissues, as has been described for GS-I (Abdul-Baki and Ray, 1971). Given the importance of hemicellulose in the primary cell wall, it is possible that this UDP-Glc transporter is essential for normal plant development.

In addition to UDP-Glc, a number of other nucleotide sugars are required for hemicellulose and pectin biosynthesis in the Golgi apparatus, and it is possible that different transporters might be involved in this process. Evi- dente supporting this idea was provided by Hayashi et al. (1988), who suggested the presence of a UDP-Xyl transporter in soybean Golgi membranes.

Transfer of Clc to Endogenous Acceptors

When UDP-Glc is inside the lumen of Golgi vesicles, Glc is transferred to polysaccharides. Our experiments in which we used 1 PM UDP-Glc and permeabilized Golgi vesicles indicate that, in contrast to what is seen in mammalian systems, UDP-Glc is not concentrated within the Golgi vesicle. It has been proposed for rat liver Golgi apparatus that a concentration step would be necessary to reach the K , of the glycosyltransferases involved in protein glycosylation (Hirschberg and Snider, 1987). Since we saw no change in the incorporation of Glc into permeabilized vesicles at 1 p~ UDP-[3H]Glc, it is possible that the K , for UDP-Glc of the glucosyltransferase would be similar or lower than the K , of the transporter. Based on this result, the transport of UDP-Glc may be the limiting step in polysaccharide biosynthesis.

It is not clear whether the initial acceptor of Glc is the elongating polysaccharide or another molecule. Dhugga et al. (1991) described a 40-kD protein located in the Golgi membrane that was transiently glucosylated and related to but distinct from GS-I. So far, we believe that nothing is known about the role of this protein in polysaccharide biosynthesis or the order of events that would lead to polysaccharide production. No topological analysis has been done regarding the activity of this protein, but according to our findings the protein domain that is glucosylated should be facing the lumen of the organelle. However, at this point we cannot rule out that this 40-kD protein has a different orientation within the Golgi apparatus.

Metabolism and Exit of the Nucleotide Moiety

When Glc is transferred, the other product of this reaction is UDP. Ray et al. (1969) found that UDP can inhibit the glucosyltransferase activity, and we have confirmed those results (data not shown). It is, therefore, important to remove UDP from the lumen of the Golgi cisternae. Our subcellular fractionation analysis showed that vesicles with UDP-Glc incorporation co-migrated with latent UDPase

activity in linear SUC gradients. Since the formation of UDP and the active site of latent UDPase are in the lumen of Golgi vesicles, the hydrolysis of UDP into UMP plus Pi can be expected, and indeed we have evidence that this reaction takes place (A. Orellana, P. Mufioz, and L. Noram- buena, unpublished data). We suggest that the physiolog- ical substrate for Golgi latent UDPase is derived from UDP-Glc and that this enzyme could be important in polysaccharide biosynthesis.

A correlation between latent UDPase and polysaccharide biosynthesis was described previously (Dauwalder et al., 1969; Ray et al., 1969; Mitsui et al., 1994), but no mechanism to account for the latent UDPase has been postulated. In yeast, the importance of a Golgi GDPase in mannosylation reactions in vivo has been well established. The disruption of the gene GDAZ, encoding for the Golgi GDPase in Sac- charomyces cerevisiae, led to a decrease in mannosylation of both proteins and lipid (Abeijon et al., 1993). Measure- ments of transport of GDP-Man into vesicles derived from the nu11 mutant indicated that GDPase plays an important role in the transport of GDP-Man (Berninsone et al., 1994). Having this evidence, we think that in plants the Golgi UDPase may play a similar role to yeast Golgi GDPase in polysaccharide biosynthesis.

Since the Golgi vesicles are a sealed compartment, both UMP and UDP need to leave the vesicles for further metabolism. Our results suggest that exit of this (or these) nucleotide(s) is coupled to UDP-Glc entrance. We therefore hypothesize that, in analogy to nucleotide sugar transporters in mammals and yeast, in plants the UDP-Glc transporter may function as an antiporter (Capasso and Hirschberg, 1984). In yeast, it was demonstrated that the antiporter uses nucleoside monophosphate rather than nucleoside diphosphate (Berninsone et al., 1994); however, at this time we cannot conclude whether the plant Golgi antiporter uses UDP, UMP, or uridine.

In our experiments we found that UDP and UMP could also promote exchange with uridine-containing nucleotides. This result does not seem to fit into the normal function of a UDP-Glc exchanger; however, a similar result was shown for the UDP-GlcNAc/UMP exchanger in rat liver Golgi vesicles, since UMP and UDP can promote exchange with luminal UMP. This phenomenon would correspond to a nonphysiological reaction of the exchanger that can be detected in the in vitro system (Waldman and Rudnick, 1990).

To our knowledge, this is the first attempt to analyze the topography of reactions leading to polysaccharide biosynthesis in the Golgi apparatus of plants. The results suggest that a nove1 UDP-Glc transporter is required for Glc-containing polysaccharide biosynthesis. Moreover, latent UDPase may play a role in this process. The use of this Golgi-vesicle preparation should help to answer a number of questions regarding the mechanism of polysaccharide biosynthesis that still remain to be resolved, such as the actual mechanism of polysaccharide biosynthesis and its regulation and the incorporation of other sugar nucleotides required for hemicellulose and pectin biosynthesis.



1594 Mufíoz et al. Plant Physiol. Vol. 1 1 2 , 1996

ACKNOWLEDCMENTS

We thank Drs. C.B. Hirschberg, E. Mandon, C. Clairmont, and M.T. Nuiiez for critica1 reading of the manuscript; Dr. M.J. Chrispeels for reading the final copy of the manuscript; a11 of the members of the Laboratory of Membranes for helpful discussions of our work; Drs. L. Cardemil and L. Corcuera for plant growth facilities; and Gloria Aravena for technical support.

Received May 6, 1996; accepted August 28, 1996. Copyright Clearance Center: 0032-0889/96/ 112/1585/ 10.

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