A 56-kDa selenium-binding protein participates in intra-Golgi protein transport.

Transport of proteins between intracellular membrane compartments is a highly regulated process that depends on several cytosolic factors. By using the well characterized intra-Golgi cell-free transport assay, we purified from bovine brain cytosol a 56-kDa protein that shows a significant transport activity. Partial sequencing of four tryptic peptides obtained from the 56-kDa protein revealed its identity to a cytosolic protein previously characterized as a selenium-binding protein, SBP56. Recombinant SBP56 expressed in Escherichia coli exhibited transport activity when added to the cell-free intra-Golgi transport. Affinity purified anti-SBP56 polyclonal antibodies specifically inhibited intra-Golgi transport in vitro. Although SBP56 is predominantly localized in the cytosol, a significant amount is associated with membranes. Subcellular fractionation showed that this protein is peripherally associated with the Golgi membrane. The experiments presented in this study indicate that SBP56 participates in late stages of intra-Golgi protein transport.

Vesicles that bud from a donor membrane compartment and then dock and fuse with an acceptor membrane mediate transport of proteins between different organelles of eukaryotic cells (1,2). This process is highly dependent on a large number of soluble and membrane proteins.
Based on the current concept of vesicular transport, budding is regulated by small GTPases and mediated by cytosolic coat proteins that assemble on the donor membrane (3,4). Coat protein (COP) 1 II vesicles mediate transport of proteins from the ER to the Golgi (5-7), whereas COPI vesicles are implicated in transport through the Golgi stack (8 -10) as well as in retrograde transport of proteins from the Golgi back to the ER (10 -12).
Targeting of a transport vesicle to its acceptor membrane is comprised of well defined steps, each involving a different set of proteins. Transport of a vesicle between distant organelles may involve the use of cytoskeletal motor proteins and cytoskeleton tracks (13)(14)(15)(16). Another set of proteins is required for tethering the transport vesicle to its acceptor organelle. For example, p115, a peripheral membrane protein that was identified and purified on the basis of its stimulatory transport activity (17), provides a linkage between COPI vesicles and the Golgi (18); Uso1p, the yeast homologue of the mammalian p115, has been suggested to act together with the small GTPase Ypt1p prior to the actual docking step (19 -22); and the Rab5 effector, EEA1 (23), has been recently shown to act as a tethering protein in early endosomal fusion together with Rabaptin-5 and the endosomal t-SNARE syntaxin 13 (24). Following tethering, docking of a vesicle at the appropriate target membrane involves the interaction between integral membrane proteins located on the vesicle, v-SNAREs, and t-SNAREs at the target membrane (25,26). The events that follow docking are under considerable debate. By using liposomes reconstituted with t-or v-SNAREs, Rothman and co-workers (27)(28)(29) showed that the v-t-SNARE complex per se fulfills the minimal requirement for fusion between two membranes. However, based on an in vitro system that reconstitutes homotypic fusion of yeast vacuoles, Ungermann and co-workers (30) deduced that the formation of the SNARE complex is only an intermediate step in the overall fusion reaction. According to this view, SNARE molecules are involved only in docking between donor and acceptor membranes, whereas another set of proteins participates in subsequent stages of the fusion process.
A variety of cell-free systems that reconstitute specific transport steps was used to identify many proteins implicated in intracellular vesicular transport. A well studied transport system that reconstitutes transport of proteins between early Golgi cisternae was originally developed by Rothman and coworkers (31). This assay measures the glycosylation of a cargo protein (vesicular stomatitis virus (VSV) G protein), present in one Golgi membrane population, by glycosyltransferase present in another stack of Golgi that does not contain the cargo protein. By using this assay, it has been shown that COPIcoated transport vesicles are formed in a reaction that utilizes a small GTPase, termed ARF1, and a complex of cytosolic coat protein termed coatomer (32). Docking and fusion in this assay require a set of membrane and cytosolic factors. NSF, SNAP, p115, GATE-16, phosphatidylinositol transfer protein ␣, and a 13 S Golgi transport complex were isolated as cytosolic factors that are essential for this assay (17,(33)(34)(35)(36)(37), apparently in mediating docking and fusion. Rab6, a Golgi-associated member of a large family of small GTPase (38,39), is also required in this cell-free transport assay, most likely at the membrane docking step (40). However, additional soluble factors are required to reconstitute intra-Golgi transport (17,35,41,42).
By using this intra-Golgi cell-free transport assay, we describe in this study the identification of a novel, soluble factor that participates in intra-Golgi transport in vitro. We have purified this transport activity 2500-fold using conventional chromatography steps, testing each fraction for its capability to stimulate intra-Golgi transport in the presence of all complementary cytosolic factors. The purification procedure described here resulted in a highly enriched fraction containing two polypeptides, identified as phosphoglucomutase (PGM) and a 56-kDa selenium-binding protein (SBP56). The transport activity was attributed to SBP56, originally discovered as a cytosolic protein that binds exogenously administered radioactive selenium (43,44). To date, however, the physiological role of SBP56 has not been elucidated. The data presented in the present study suggest that SBP56 participates in late stages of intra-Golgi transport.

Solutions
Most solutions used in this work are coded as follows: T, 25

Preparation of Cytosolic Factors
Bovine brain and rat liver cytosols were prepared as described by Malhotra et al. (8) and Waters et al. (17), respectively. Fraction ␤ was prepared as described previously (35). P115 and Rab-GDI were purified from bovine liver cytosol as described previously (17,45).

I␣-dependent Intra-Golgi Transport Assay
The standard intra-Golgi transport was performed as described previously (31). The I␣-dependent intra-Golgi transport assay (25 l) contained 0.4 Ci of UDP-[ 3 H]N-acetylglucosamine, 5 l of a 1:1 mixture of donor and acceptor Golgi membranes (2-3 g of protein), 2.5 l (10 g) of ␤ cytosolic fraction, 2.5 l (6 g) of II␣ cytosolic fraction, 0.25 l (6.25 ng) of His 6 NSF, 0.25 l (30 ng) of His 6 ␣SNAP, 1.25 l (0.5 g) of p115, 10 M palmitoyl-coenzyme A, and ATP-and UTP-regenerating systems. To test for the transport activity of I␣, different cytosolic fractions were added to the I␣-dependent assay mixture. The transport reactions were incubated at 30°C for 2 h. [ 3 H]N-acetylglucosamine incorporated into VSV-G protein was determined as described previously (35). Each of the transport assay experiments shown in this study represents at least three independent assays performed in duplicate. Background (200 -310 cpm) detected in the absence of cytosolic fraction was subtracted from all experiments.
For the glycosylation assay, "wild-type donor" membranes were prepared as described by Taylor et al. (46). Briefly, Golgi membranes were isolated from wild-type CHO cells infected with VSV, after which the isolated wild-type donor membranes were treated with NEM (1 mM) for 15 min on ice, at which time DTT (2 mM) was added to quench any remaining NEM. The glycosylation assay was performed under identical conditions described for either the standard transport assay or the I␣-dependent assay described above.

Purification of I␣
Ammonium Sulfate Precipitation-Starting with 8.4 g of bovine brain cytosol, the cytosol concentration was adjusted to 12 mg/ml by dilution with 50 KT␤mM, pH 7.4. Ammonium sulfate was slowly added while stirring until a final concentration of 40% saturation at 4°C was reached. The solution was stirred for additional 30 min and centrifuged in a Sorvall GS3 rotor at 8,500 rpm for 30 min. The pellet was resuspended in 120 ml of 50 KT␤mGM and dialyzed against 50 KT␤mM, and the insoluble material was removed by ultracentrifugation in a Beckman 45Ti rotor at 35,000 rpm for 1 h. This material was designated AS precipitate.
Fast Flow Q Chromatography-The AS precipitate was adjusted to 5 mg/ml with 100 KT␤mGM, pH 7.4, and loaded at 5 ml/min onto a 300-ml Fast-flow Q column equilibrated with 100 KT␤mGM. The column was washed with 400 ml of equilibration buffer, and the flowthrough material (1.2 mg/ml) was collected. This material was designated ␣. The bound material of this column was used for preparing the cytosolic fraction ␤.
Fast Flow CM Chromatography-The ␣ material was dialyzed against NaP i ␤mM, pH 6.8, centrifuged in a Sorvall GS3 rotor at 8,500 rpm for 10 min, and then loaded at 5 ml/min onto a 150-ml Fast-flow Carboxyl Methyl (CM) column equilibrated in NaP i ␤mGM, pH 6.8. The column was washed with 150 ml of the same buffer, and the flowthrough material, designated I␣, was collected. This material had a protein concentration of 0.7 mg/ml. Recently, a 16-kDa novel protein (GATE-16) involved in transport was purified from the bound material of this column (35,59). The bound material containing p16 was used for preparing the cytosolic fraction II␣.
Isoelectric Precipitation-The I␣ was dialyzed extensively against NaP i ␤mM, pH 5.0, and centrifuged in a Sorvall GS3 rotor at 8,500 rpm for 15 min at 4°C. The pellets were discarded, and supernatants were pooled. This material, designated I␣pH 5.0, had protein concentration of 0.085 mg/ml.
Mono-S Chromatography-The conductivity of I␣pH 5.0 was adjusted to that of NaP i ␤mGM buffer, pH 5.0, by dilution with the same buffer to a final volume of 750 ml. I␣pH 5.0 was then loaded at 2 ml/min onto a 20-ml Mono-S column (fast performance liquid chromatography) and equilibrated in NaP i ␤mGM, pH 5.0. The column was washed with 24 ml of the equilibration buffer, and proteins were eluted with a 170-ml gradient of 0 -500 mM KCl in NaP i ␤mGM buffer (2.94 mM/ml gradient). Fractions of 4 ml were collected throughout, and 10-l samples were tested for their transport activity using the I␣-dependent transport assay. Fractions exhibiting significant intra-Golgi transport activity, eluted at about 150 -350 mM KCl, were pooled and collectively designated I␣MS. This material had protein concentration of 0.093 mg/ml.
Phenyl-Superose Chromatography-The conductivity of I␣MS was adjusted to 1500 mM KCl by the addition of solid KCl, and the pH was adjusted to 7.4 by Tris base titration. I␣MS was then loaded at 0.3 ml/min onto 1-ml phenyl-Superose column (fast performance liquid chromatography) equilibrated in 1500 KT␤mGM. The column was washed with 8 ml of equilibration buffer, and proteins were eluted with a 20-ml decreasing gradient of 1500 to 0 mM KCl in KT␤mGM (Ϫ37.5 mM/ml gradient), followed by 20-ml elution of 0 KT␤mGM. Fractions of 1 ml were collected throughout, and 10-l samples were tested for their transport activity using the I␣-dependent transport assay. Fractions showing transport activity, eluted at 80 -0 mM KCl, were pooled and collectively designated I␣PS. This material had protein concentration of 0.02 mg/ml.
Hydroxylapatite Chromatography-I␣PS was dialyzed against NaP i ␤mGM, pH 6.8, and loaded at 2 ml/min onto ϳ1-ml hydroxylapatite column equilibrated in the dialysis buffer. The column was washed with 5 ml of equilibration buffer and then subjected to a 10-ml phosphate gradient from 10 to 225 mM NaP i ␤mGM (21.5 mM/ml gradient), followed by a steeper phosphate gradient of 225-400 mM NaP i ␤mGM (35 mM/ml gradient). Fractions of 0.5 ml were collected throughout, and 10-l samples were tested for their transport activity using the I␣-dependent transport assay. Fractions showing transport activity, eluted at about 15-50 mM NaP i , were pooled and collectively designated I␣HA. This material had protein concentration of 0.0045 mg/ml.

Tryptic Digestion, Separation, and Sequencing of Peptides
To obtain the amino acid sequence of the proteins found in I␣HA, ϳ10 g were separated on 10% SDS-PAGE. Coomassie Brilliant Bluestained protein bands were excised, digested in situ with trypsin, and the resultant fragments were separated by reverse phase-high pressure liquid chromatography. Peptides were sequenced with automated sequencer at the MicroSequencing Unit, the Protein Center, the Technion, Israel.

SDS-PAGE and Immunoblot Analysis
For most analyses, electrophoresis was performed on 10% polyacrylamide gels (8 ϫ 10 cm), after which proteins were blotted onto nitrocellulose. Antibody incubations were carried out in 1% bovine serum albumin and 0.1% Tween 20 in phosphate-buffered saline. The source of antibodies and dilutions for Western blots are indicated specifically for each assay. All antibodies were reacted either by peroxidase-conjugated anti-mouse or anti-rabbit, followed by enhanced chemiluminescence detection. Proteins were visualized on polyacrylamide gels by staining with Coomassie Brilliant Blue.

Expression and Purification of Recombinant SBP56
The full-length SBP56 cDNA in pRc/CMV plasmid was received as a gift from the laboratory of Dr. P. R. Harrison, the Beatson Institute for Cancer Research, Glasgow, UK. The full-length SBP56 cDNA was subcloned using HindIII into pQE11 (Qiagen) plasmid containing His 6 tag in the N terminus. Escherichia coli JM109 (DE3), transformed by the pQE11-SBP56 construct, was grown in 12 liters of Luria-Bertani (LB) broth containing 100 g/ml ampicillin and 50 g/ml kanamycin at 37°C to an A 600 of 0.90, induced with 1 mM IPTG for 4 h, and centrifuged in Sorvall GS3 rotor at 8,500 rpm for 15 min. The bacterial pellet was resuspended in a breaking buffer containing 25 mM Tris, pH 7.5, 250 mM KCl, 10 mM 2-mercaptoethanol, 20 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, 0.5 g/ml leupeptin, 2 g/ml aprotinin, and 2 M pepstatin A. Resuspended bacteria cells were lysed by the French press technique and clarified by centrifugation at 35,000 rpm (119,000 ϫ g av ) for 90 min, using 45Ti rotor (Beckman). Clarified supernatant was loaded over a nickel-nitrilotriacetic acid (Ni-NTA)agarose column (Qiagen) and washed with 50 ml of breaking buffer, and His 6 -SBP56 protein was eluted by linear gradient of 20 -300 mM imidazole. Fractions of 5 ml, containing recombinant SBP56 (rSBP56), were collected, dialyzed against 25 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT, and 10% (v/v) glycerol, and stored in aliquots at Ϫ80°C until use.

Affinity-purified Anti-SBP56 Antibodies Preparation
His 6 -tagged SBP56 was purified as described above and used to raise polyclonal antibodies in rabbits by the animal service unit at the Weizmann Institute of Science. Specific anti-SBP56 polyclonal antibodies were affinity-purified on a cyanogen bromide-activated Sepharose column coupled to His 6 -SPB56. Eluted fractions containing antibodies were pooled and concentrated on Centricon-30 filters (Amicon). The buffer was then changed to phosphate-buffered saline using 5-ml Hi-Trap Sephadex G-25 column (Amersham Pharmacia Biotech). Aliquots were stored at Ϫ20°C until use.

Subcellular Fractionation of SBP56
Rat liver lysates were fractionated over sucrose gradients as described previously (47) with several modifications. Briefly, fresh rat livers (25 g) were washed with a buffer containing 25 mM Tris, pH 7.4, and 320 mM sucrose. The tissue was then homogenized with a Teflon homogenizer in 150 ml of ice-cold lysis buffer (0.1 M KP i , pH 6.8, 0.5 M sucrose, 5 mM MgCl 2 , 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.5 g/ml leupeptin, 2 g/ml aprotinin, and 2 M pepstatin A). The homogenate was centrifuged in an SS-34 rotor (Sorvall) at 1360 ϫ g av for 10 min. The post-nuclear supernatant was decanted, and 18 ml were layered over each of four 0.86/1.25 M sucrose step gradients (10 ml each step) in SW-28 centrifuge tubes (all sucrose solutions were buffered with 0.1 M KP i , pH 6.8, 5 mM MgCl 2 ). Gradients were centrifuged at 4°C in an SW-28 rotor (Beckman) at 25,000 rpm (82,700 ϫ g av ) for 90 min. The 0.86/1.25 M interface was adjusted to 1.6 M sucrose by 2 M sucrose solution, and 10 ml were loaded onto each of two SW-28 centrifuge tubes. This layer was overlaid with 1.25, 1.0, 0.86, and 0.5 M sucrose layers (each of 7 ml). The gradients were centrifuged at 4°C in a SW-28 rotor (Beckman) at 25,000 rpm (82,700 ϫ g av ) for 2.5 h with slow acceleration and deceleration. All fractions from the second gradient were subjected to 12% SDS-PAGE. Fractionated proteins were subjected to Western blot analysis with affinity-purified anti-SBP56 (1: 5000 dilution), anti-GOS28 monoclonal antibodies (1:20 dilution), and anti-PDI monoclonal antibodies (SPA-891 1:1000 dilution).

RESULTS
Establishing the I␣-dependent Intra-Golgi Transport Assay-Several cytosolic and peripheral membrane proteins involved in vesicular transport were previously identified using an in vitro system that reconstitutes intra-Golgi transport (2). In this system the requirement for crude cytosol can be replaced by a complementation assay in which a signal is observed only upon the addition of the protein of interest in the presence of saturating levels of all known cytosolic factors. Following this rationale, we have recently developed a purification scheme for the isolation of a novel cytosolic transport factor, GATE-p16 (35,59). In the process of cytosol fractionation, we identified two crude fractions, I␣ and ␤, that are required for a successful reconstitution of intra-Golgi transport (see scheme in Fig. 1A). To focus on the activity found in fraction I␣, we developed a specific I␣-dependent transport assay in which NSF, ␣SNAP, p115, fraction II␣, and fraction ␤ are required to replace crude cytosol (Fig. 1B). However, these cytosolic proteins were not sufficient to reconstitute intra-Golgi transport, unless fraction I␣ was added. The reconstitution brought about by I␣ (Fig. 1B, right panel) was somewhat less efficient than transport obtained with crude cytosol (Fig. 1B,  left panel). The I␣-dependent transport assay was found sensitive to NEM, and the transport activity could be partly restored with recombinant NSF, similar to the assay reconstituted with crude cytosol (Fig. 1C). Hence, it appears that I␣ acts as part of the known transport machinery. This assay was therefore used in the present study to purify and characterize the active factor present in I␣.
I␣ Is Involved in Late Stages of Transport-Vesicular transport was shown to be blocked by GTP␥S, a nonhydrolyzable analogue of GTP (48). GTP␥S fails to block the observed signal  KCl). B, increasing amounts of crude rat brain cytosol (left) or I␣ (right) were added to the standard or to the I␣-dependent transport assays, respectively (see "Experimental Procedures"). C, CHO Golgi membranes were either kept on ice or treated with 1 mM N-ethylmaleimide (NEM) for 15 min and quenched for 10 min with 2 mM DTT, prior to their addition to the standard transport assay (left) or to the I␣-dependent transport assay (right). Reactions were carried out in the presence or absence of N-ethylmaleimide-sensitive factor (NSF). [ 3 H]GlcNAc incorporation into VSV-G protein was measured after 2 h incubation at 30°C. when (i) either the small GTPase, an ADP-ribosylation factor, or coatomer is removed from the cytosol (42,46), or (ii) when their function is blocked by brefeldin A (BFA) (49), conditions that uncouple fusion from budding (42). Thus, sensitivity of the transport assay to GTP␥S and BFA indicates whether the obtained signal results from a fusion reaction coupled to vesicles budding. As shown in Fig. 2, GTP␥S inhibited transport reconstituted with crude cytosol, an inhibition that could be reversed by BFA. In contrast, the I␣-dependent assay was stimulated by GTP␥S and remained resistant to BFA (Fig. 2), suggesting that I␣ is involved in the uncoupled fusion process, namely docking or fusion rather than budding. The stimulation observed in the presence of GTP␥S might be attributed to activation of other GTP-binding proteins involved in this process.
Purification of Fraction I␣-In the first step of cytosol fractionation, 40% ammonium sulfate was used to differentially precipitate proteins from bovine brain cytosol. The ammonium sulfate precipitate was chromatographed on a Q-Sepharose anion exchange column followed by chromatography on a CM-Sepharose cation exchange column to which the active fraction did not bind (Fig. 1A). The CM-Sepharose unbound material (I␣) was then fractionated by isoelectric precipitation, Mono-S cation exchange, phenyl-Superose chromatography, and hydroxylapatite chromatography (for details see Table I and "Experimental Procedures").
Active fractions eluted from the phenyl-Superose column (I␣PS) were chromatographed on a hydroxylapatite column. Fractions eluted from this column by relatively low (50 mM) sodium phosphate concentration showed transport activity (Fig. 3A). Fractions 10 -12 (I␣HA) were pooled and separated on SDS-PAGE, yielding two major polypeptides of 56 and 65 kDa (Fig. 3B). As shown in Fig. 3C, increasing amounts of I␣HA, pure I␣, stimulated intra-Golgi transport, which became saturated at about 20 g/ml. Activity levels at each step of the purification process and protein yields are shown in Table I. The elevated total activity at the first step of the purification is presumably due to extraction of components that interfered with the I␣-dependent assay. In addition, at early stages of the purification the transport assay is sensitive to more than one type of transport factor. Hence, the specific activity significantly increased only at the last four steps of purification. A typical purification protocol yielded approximately 50 g of I␣ protein. A sample (4.5 g) is shown in Fig. 3B. Altogether, there was 2500-fold increase in the specific activity with a 1.5% activity yield ( Table I).
Identification of I␣-To identify the 56-and 65-kDa polypeptides, the corresponding bands were excised from the gel, and the amino acid sequence of four tryptic peptides obtained from each band was determined (Fig. 3B). All amino acid sequences of the tryptic peptides obtained from the 65-kDa polypeptide band were identical to phosphoglucomutase (PGM). This enzyme catalyzes the transfer of Glc-1-P to Glc-6-P and enables glycogen building blocks to enter the glycolytic pathway. A commercially available PGM (Roche Molecular Biochemicals) was tested for its ability to complement the I␣-dependent transport assay. Although fully active as PGM, the enzyme had no effect on the I␣-dependent transport assay (data not shown). We thus focused on the 56-kDa protein, the major band in the pure I␣ fraction. All four tryptic peptides obtained from that band (Fig. 3B) had an identical amino acid sequence to the mouse 56-kDa selenium-binding protein (SBP56) (44). By using specific anti-SBP56 antibodies we show that SBP56 is predominantly present in the cytosolic fraction I␣ (Fig. 3D). Only a small amount of SBP56 was detected in the cytosolic fraction ␤, which may account for the low but significant background detected in the I␣-dependent assay.
SBP56 Is Implicated in the Cell-free Intra-Golgi Transport Assay-To determine whether SBP56 is directly involved in intra-Golgi transport, we cloned and expressed a recombinant form of this protein and examined its activity in the I␣-dependent transport assay. For that purpose, rat cDNA of SBP56 was subcloned into a pQE11 vector to produce a protein tagged with six histidine residues at its N terminus. The protein was expressed in E. coli and purified on a Ni-NTA-agarose column (Fig. 4A). The recombinant SBP56 (rSBP56) was tested for its ability to replace pure I␣ fraction in the I␣-dependent transport assay. Increasing amounts of rSBP56 significantly stimulated the transport activity; heating the rSBP56 to 65°C for 20 min abolished this activity (Fig. 4B). The low activity of either purified I␣ or rSBP56 in comparison with the crude I␣ pool suggests that I␣ contains additional yet unidentified transport factors.
To assess the involvement of the endogenous SBP56 in the intra-Golgi transport activity, we reconstituted the transport assay with crude cytosol. In this system, addition of increasing amounts of affinity-purified anti-SBP56 antibodies inhibited up to 85% of the transport activity (Fig. 4C). When rSBP56 was preincubated with the antibodies, the transport activity was restored to almost its maximal level (Fig. 4C). Preimmune immunoglobulins used for control did not affect the transport assay, indicating specific inhibition exerted by the anti-SBP56 antibodies. Maximal inhibition of transport was observed only FIG. 2. I␣ participates in late stages of transport. Bovine brain cytosol (15 g) was assayed in the standard transport assay (left), and I␣ (4 g) was assayed in the I␣-dependent transport assay (right). GTP␥S (25 M) or BFA (150 M) were added as indicated. A value of 100% corresponds to 1900 cpm in the standard transport assay and to 1100 cpm in the I␣-dependent transport assay. when the anti-SBP56 antibodies were added upon the initiation of transport.
To test further the involvement of SBP56 in intra-Golgi transport, the cytosol was depleted of SBP56 by specific anti-SBP56 antibodies. Affinity-purified anti-SBP56 antibodies, covalently coupled to protein-A beads, precipitated more than 90% of the SBP56 from the cytosol (Fig. 5A). The removal of SBP56 from the cytosol was accompanied by about 72% reduction in intra-Golgi transport (Fig. 5B); addition of rSBP56 to the depleted cytosol significantly recovered the transport activity (Fig. 5B). These experiments provide further support for the involvement of SBP56 in intra-Golgi transport.
We took two independent approaches to exclude the possibility that the stimulation of the assay signal by SBP56 resulted from enhanced glycosylation activity of N-acetylglu-cosamine (GlcNAc) transferase rather than stimulation of transport. We first utilized a glycosylation assay originally developed by Melancon and co-workers (46). Briefly, Golgi membranes were isolated from wild-type CHO cells infected with VSV. In these membranes the VSV-G protein and GlcNActransferase I are present in the same compartment. In this system the transfer of radiolabeled GlcNAc to VSV-G protein is independent of membrane fusion (46). Hence, the cytosol is not required to generate the assay signal, which remains resistant to the alkaline reducing agent NEM (known to block the standard transport assay). As shown in Fig. 6A, rSBP56 significantly stimulated the I␣-dependent transport assay, whereas addition of rSBP56 to the I␣-dependent glycosylation assay (see "Experimental Procedures") had no effect nor did the cytosolic endogenous SBP56 (data not shown). Furthermore, anti-SBP56 antibodies, which inhibit up to 90% of the standard transport FIG. 3. A 56-kDa selenium-binding protein accounts for the I␣ transport activity. A, in the last step of I␣ purification, I␣PS was loaded onto 1-ml hydroxylapatite column (see "Experimental Procedures"). Samples from each fraction were then tested for transport activity in the I␣-dependent transport assay. B, the protein pool (fractions 10 -12) eluted from the hydroxylapatite column was separated on 10% SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Amino acid sequences of peptides obtained from phosphoglucomutase (PGM) and selenium-binding protein of 56-kDa (SBP56) are shown. C, increasing amounts of the pure I␣ pool (I␣HA) added to the I␣-dependent transport assay. D, Western blot analysis with anti-SBP-56 antibodies of bovine brain cytosol (60 g), fraction I␣ (2.4 g), fraction ␤ (15 g), and pure p115 (0.5 g).

FIG. 4. Intra-Golgi transport activity of recombinant SBP56. A,
SBP56 was cloned into a pQE11 and expressed in E. coli to create a six histidine-tagged recombinant protein, as described under "Experimental Procedures." E. coli protein profile was obtained in the absence (lane 1) or presence (lane 2) of 1 mM isopropyl-1-thio-␤-D-galactopyranoside, and proteins eluted from a Ni-NTA column (lane 3) were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining. B, purified His 6 -SBP56, either kept on ice (filled triangles) or heat-inactivated at 65°C for 20 min (open circles), was added to the I␣-dependent intra-Golgi transport assay. C, affinity-purified polyclonal antibodies directed against SBP56 or pre-immune antibodies were added to the standard transport assay as indicated. For the control assay, anti-SBP56 antibodies were incubated with rSBP56 and then were added to the transport assay. assay, did not affect the signal generated in the glycosylation assay (Fig. 6A).
To exclude further the possibility that SBP56 is involved in glycosylation, the rate-limiting step of the standard transport assay (50), we have performed a kinetic experiment in which we compared the effects of GTP␥S and anti-SBP56 antibodies (Fig. 6B). In the experiment described in Fig. 6B, the transport assay was either terminated at different time points by ice or each of the inhibitors, anti-SBP56 or GTP␥S, was added at the indicated time points, after which the reaction was allowed to proceed for a 2-h incubation period. Resistance of the reaction at a distinct time interval may also indicate a sequence of events of the intra-Golgi transport assay. Standard samples, receiving only buffer, were further incubated at 30°C until the end of the 2-h incubation period and served as control (100% transport). Both inhibitors, when added at the onset of the reaction, brought about 90% inhibition of transport. The reaction became resistant to the anti-SBP56 antibodies when these were added 40 -60 min after transport was initiated. This indicates that SBP56 is acting prior to the glycosylation step represented by the ice samples. Furthermore, the transport became resistant to the anti-SBP56 antibodies after the GTP␥S inhibition, further supporting the notion that SBP56 operates downstream to vesicle uncoating.
SBP56 Acts Downstream to Rab Proteins-Small GTPases of the Rab family were shown to function in intra-Golgi transport in vitro (40,45,51). Rab-GDP dissociation inhibitor (GDI) added in micromolar amounts resulted in removal of most Rab proteins from intracellular membranes (52) thus causing inhibition of intra-Golgi transport (45). It has been recently demonstrated that Rab-GDI specifically blocks vesicle docking but not their fusion with the target membrane (22). In the present study, we utilized Rab-GDI to determine the stage at which SBP56 is active. We first tested whether the inhibition of intra-Golgi transport observed by either Rab-GDI or anti-SBP56 antibodies was reversible. To this end, we performed a two-stage transport assay in which Golgi membranes were first treated with different inhibitors for 20 min under standard transport conditions and then reisolated, washed, and tested for transport activity in the presence of fresh cytosol. As shown in Fig. 7A, addition of fresh cytosol to Golgi membranes that were previously incubated with either anti-SBP56 or GDI recovered up to 80% of the original intra-Golgi transport. Fig. 7B describes the results of a two-stage transport assay in which Golgi membranes were first incubated with the indicated inhibitor for 20 min and then isolated, washed, and incubated with fresh cytosol in the presence or absence of the indicated inhibitor. Anti-SBP56 antibodies were found to inhibit the transport activity of Golgi membranes that were pretreated with Rab-GDI suggesting that Rab proteins act earlier than SBP56. In a reciprocal experiment we demonstrated that Rab-GDI did not inhibit the transport activity of Golgi membranes that were pretreated with anti-SBP56 antibodies (Fig. 7B). These experiments suggest that SBP56 acts downstream to Rab proteins in cell-free intra-Golgi transport, further supporting its role in late stages of the transport.
SBP56 Is Associated with Golgi Membranes-Following tissue homogenization, most SBP56 was found in the soluble FIG. 5. Immunodepletion of SBP56 reduces intra-Golgi transport activity. A, Western blot analysis of SBP56-depleted rat liver cytosol. Rat liver cytosol was immunodepleted of SBP56 using affinitypurified polyclonal antibodies and analyzed by Western blotting with the same antibodies. Lane 1, rat liver cytosol (3 g); lane 2, rat liver cytosol incubated with protein A beads (2.6 g); lane 3, SBP56 rat liver cytosol treated with protein A beads coupled to anti-SBP56 antibodies (2.35 g). B, in vitro intra-Golgi transport assay was performed with different cytosolic fractions (12 g) as follows: lane 1, rat liver cytosol; lane 2, rat liver cytosol incubated with protein A beads; lane 3, rat liver cytosol treated with protein A beads coupled to anti-SBP56 antibodies; and lane 4, rat liver cytosol treated with protein A beads coupled to anti-SBP56 antibodies in the presence of 350 ng of rSBP56.
FIG. 6. SBP56 specifically activates intra-Golgi transport and not GlcNAc-transferase I. A, anti-SBP56 antibodies (0.6 g) or rSBP56 (0.4 g) were added to the standard cell-free transport assay or to the glycosylation assay (see "Experimental Procedures"). The inhibitory effect of the anti-SBP56 antibodies was tested in assays reconstituted with crude cytosol as indicated, whereas the stimulatory effect of rSBP56 was tested in a I␣-dependent assay (see "Experimental Procedures"). The Golgi membranes used for the glycosylation reaction were pre-treated with 1 mM NEM (see "Experimental Procedures"). 100% corresponded to 2,200 or 1,440 cpm in the standard or the glycosylation assays, respectively. B, a standard intra-Golgi transport assay was carried out at 30°C for 2 h. At the indicated time points, anti-SBP56 antibodies (1 g, filled squares) or GTP␥S (50 M, open squares) were added, and the reaction was terminated after 2 h. The progression of transport in the absence of inhibitors was measured by transferring samples to ice at the indicated time points (open circles). fraction with only a small fraction (about 10%) associated with the membranes (data not shown). To determine the subcellular localization of the membrane-bound SBP56, rat liver post-nuclear supernatant was fractionated on two successive equilibrium density sucrose gradients. Membranes accumulated at the 0.86/1.25 M sucrose interface were collected, adjusted to 1.6 M sucrose, and loaded onto the bottom of a second sucrose gradient. Anti-SBP56 polyclonal antibodies, anti-GOS28 (a Golgi-specific v-SNARE (53,54)), or anti-PDI (an ER-resident protein) monoclonal antibodies were applied to Western blots of the different fractions obtained from the second sucrose gradient. SBP56 migrated with membranes corresponding to the Golgi apparatus (1.0 -1.25 M sucrose), and, to a lesser extent, with membranes fractionated in 1.25-1.4 M sucrose where most of the ER was found (Fig. 8A). Soluble factors such as tubulin and actin detected in the cytosolic fraction were not present in the sucrose gradient fractions corresponding to the ER and the Golgi (data not shown), indicating that SBP56 was specifically associated with these membranes.
The finding that only a very small portion of SBP56 was bound to membranes suggests that this protein is only transiently associated with the Golgi membranes. It appears that SBP56 associates well with the membranes at 60 mM KCl (Fig.  6B), conditions that are optimal for intra-Golgi transport in vitro. Attempts to dissociate all SBP56 from the membranes with increasing concentrations of salt (up to 1 M KCl) have failed, whereas treatment with 100 mM sodium carbonate, pH 11.5, removed most SBP56 from the membranes (Fig. 8B). Treating membranes with 1% Triton X-100 completely shifted SBP56 to the soluble fraction (Fig. 8B). These experiments imply that part of SBP56 is peripherally associated with the membrane. By using isolated CHO Golgi membranes and recombinant SBP56, we found that this protein specifically binds to membranes although the mechanism is still unknown (data not shown). DISCUSSION We have used the well characterized cell-free assay that reconstitutes intra-Golgi transport to isolate and purify a novel 56-kDa protein from bovine brain cytosol and demonstrated its significant transport activity. The pure protein is identical to a selenium-binding protein of 56 kDa (SBP56), which was previously isolated as a protein that binds selenium (43,44), but its physiological function has not yet been elucidated. We present here several lines of evidence that implicate SBP56 in vesicular transport: (i) the endogenous protein was isolated on the basis of a functional transport assay; (ii) rSBP56 expressed in E. coli is active in the cell-free transport assay; (iii) antibodies directed against SPB56 specifically block the transport assay; (iv) cytosol depleted of SBP56 is significantly less active in stimulating intra-Golgi transport; and (v) the membrane pool of SBP56 is associated primarily with the Golgi. In addition, we have demonstrated that SBP56 is directly involved in the FIG. 7. SBP56 is acting downstream to Rab proteins. A, intra-Golgi transport was reconstituted under standard conditions with Golgi membranes and crude CHO cytosol, in the presence of 1 g of GDI or 0.5 g of anti-SBP56 antibodies throughout the transport reaction (black bars), or for 30 min at 30°C. Membranes were then isolated, washed, and incubated for another 2 h at 30°C with fresh cytosol (gray bars). 100% corresponded to 1400 cpm. B, Golgi membranes were incubated with 1 g of GDI (top panel) or 0.5 g of anti-SBP56 (bottom panel) as described above, re-isolated, washed, and incubated with fresh cytosol in presence of the indicated inhibitors. transport process in vitro rather than in affecting the assay signal by stimulating the enzymatic activity of GlcNAc transferase I.
Pure I␣ or rSBP56 significantly stimulates the cell-free transport assay, although to a lesser extent than the crude I␣ cytosolic fraction. This indicates the existence of other transport factors in the I␣ pool. Evidence for such factors came from our fractionation experiments, in which we could detect low transport activities that were independent of SBP56. 2 Attempts to characterize further these activities have proven extremely difficult.
SBP56 is a highly conserved protein with homologues found in nematodes, plants, and mammals. Yet, the amino acid sequence of SBP56 shows no significant homology to any yeast open reading frame. This is unusual since most transport factors are highly conserved between yeast and mammals. The yeast genome may carry a functional homologue of SBP56 that fulfills its function. Alternatively, SBP56 may function as a regulator of a particular step in transport that is unregulated in yeast, or its control is SBP56-independent.
SBP56 was originally discovered as a cytosolic protein that binds selenium (44). This protein is highly expressed in liver, lung, colon, and prostate, and it has been suggested that its expression level correlates with low frequency of cancer in these tissues (55). Yet the exact physiological role of this protein has not been characterized. Furthermore, neither the mode by which selenium binds to SBP56 nor the functional significance of selenium binding is known. Here we present data that implicate SBP56 in vesicular transport. We could not determine any correlation between selenium binding and the activity of this protein in the transport assay. It may well be that this protein has more than one physiological role.
It has been suggested that Rab proteins are required upstream to the SNARE complex formation (20,56), presumably after NSF and SNAP (30,57). Furthermore, using the yeast in vitro system it has been recently demonstrated that Rab proteins are required for docking of COPII vesicles to the Golgi membrane and not for fusion (22). Based on the two-stage experiment described in Fig. 7, we propose that SBP56 is active downstream of the Rab proteins. We therefore propose that SBP56 is involved in regulating docking or fusion. Considering the recent findings by Rothman and colleagues (27), who demonstrated that SNAREs comprise the minimal core machinery that drives fusion between vesicle and its target, we suggest that SBP56 functions along with other soluble factors to accelerate and regulate this process.
To date, several cytosolic factors were isolated based on their activity in the intra-Golgi cell-free transport assay. These include NSF and SNAP, which are known to act in regenerating and priming the fusion machinery (25,57); p115, a trimeric factor that bridges between COPI vesicles and the Golgi (18); GATE-16 (35), which functions together with NSF and SNAP in keeping the Golgi SNAREs active (59); PITP␣ (36); and an S 13 hetero-oligomeric complex that functions at late stages of the intra-Golgi transport (37). Rab proteins that cycle between the membrane and the cytosol (58) are not required in the cytosolic component of the cell-free transport assay. This is most probably due to a substantial pool of active Rabs in the membrane fraction. Here we describe SBP56 as another soluble protein involved in transport. We present evidence that this protein may be required at late stages of transport, namely docking and fusion. At present, however, we cannot determine the exact stage at which SBP56 is required or whether it mediates the activity of any of the known transport factors. The challenge in the near future would be to reconstitute intra-Golgi transport in vitro with a well defined set of cytosolic and peripheral membrane proteins.