Effects of Brefeldin A on the Endocytic Route REDISTRIBUTION OF MANNOSE 6-PHOSPHATE/INSULIN-LIKE GROWTH FACTOR I1 RECEPTORS TO THE CELL SURFACE*

The effect of brefeldin A (BFA) on the trafficking of the mannose 6-phosphate/insulin-like growth factor I1 receptor within the endocytic route was analyzed. Treatment with BFA induced a redistribution of the receptor to the cell surface and increased both the binding and internalization of ligands 2-4-fold. The effect of BFA was dose- and time-dependent and re- versible. Determinations of transport rates showed that BFA increases the internalization rate and the externalization rate of the receptor. This implies that the higher surface concentration is due to higher con- centrations of receptor at the intracellular sites from where they recycle to the cell surface. The effect of BFA was additive to the redistribution induced by insulin-like growth factors I and I1 and was observed in all human and rodent cell lines analyzed. BFA increased also the cell surface expression of the M, 46,000 mannose 6-phosphate receptor but not of the transferrin receptor. The results indicate that BFA interferes with the transport of mannose 6-phosphate receptors and affects the endocytosis of lysosomal en- zymes by increasing the number of receptors available for recycling to the cell surface.

Q To whom correspondence should be addressed. pathway, we studied the cellular distribution of the mannose 6-phosphate/insulin-like growth factor I1 (Man-6-P/IGF 11) receptor. This receptor is primarily localized in the TGN, late endosomes and at the plasma membrane (10,11). Receptors in these compartments are in equilibrium (12)(13)(14). In this study we report on the effects of BFA on the trafficking of the Man-6-P/IGF I1 receptor between the cell surface and internal membranes.
BFA leads to a specific and reversible redistribution of Man-6-P/IGF I1 receptors to the cell surface, which is ascribed to an increase of the receptor pool available for the recycling to the plasma membrane. A similar redistribution to the cell surface was seen for the M , 46,000 mannose 6phosphate receptor (MPR 46), which shares with the Man-6-P/IGF I1 receptor the recycling from endosomes to the Golgi as well as to the plasma membrane (1 1).

MATERIALS AND METHODS
Pentamannosyl 6-o-phosphate-substituted bovine serum albumin (Man-6-P-Man4-BSA) was prepared from Hansenulu holstii phosphomannan, provided by Dr. M. Slodki (United States Dept. of Agriculture, Northern Regional Research Center, Peoria, IL) as described (15). Man-6-P (disodium salt) and human transferrin were obtained from Sigma and Pansorbin from Calbiochem. Percoll was purchased from Pharmacia LKB Biotechnology Inc. Recombinant insulin-like growth factors (IGF) I and I1 were a kind gift by Dr.
Internalization of "'I-Labeled Arylsulfatase A-The internalized 12sII-labeled arylsulfatase A, which binds to more than 85% to a Man-6-P/IGF I1 receptor matrix in a Man-6-P dependent manner, was determined by incubation of fibroblasts for 60 min at 37 "C. The cells were chilled to 4 "C, washed, and the cell surface-bound ligand displaced by Man-6-P. The cells were solubilized in 1 N NaOH, and the cell-associated radioactivity referred to cell protein (19). The presence of 5 mM Man-6-P during the incubation period inhibited the endocytosis by 97%. Subcellular fractionation of internalized 1251-arylsulfatase A was performed on Percoll density gradient centrifugation (20).
Binding and Uptake of '?'I-Labeled Receptor Antibodies-The binding of "'I-labeled 2C2 antibodies was measured as described (15). For the internalization assay the fibroblasts were pretreated with BFA and then incubated at 37 "C for the indicated times with the "' 1labeled antibodies 21D3 or 2C2 in MEM containing 20 mM Hepes, pH 7.2, 0.1% BSA. After washing the cells with ice-cold Hanks' solution the cells were solubilized in 1 N NaOH, and cell-associated radioactivity was referred to cell protein.
Other Methods-Hep G2 cells that were used for immunoelectron microscopy were fixed in 1% acrolein and 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h at 0 "C. Subsequent preparation of cryosections and immunogold labeling of Man-6-P/IGF I1 receptors were performed as described (23). Autoradiograms and fluorograms were quantified using a laser scan densitometer (Ultroscan XL, LKB, Bromma). Alternatively the polypeptide bands were excised from the gel and either directly counted in a y counter (Berthold, bf 5300) or after solubilization (Tissue-sol-Roth M) by p-scintillation spectrometry (Packard Tri Carb 1900 TR). For the evaluation of K D and B,,, for Man-6-P-Man4-BSA the nonlinear regression method by Scatchard was used (24).

RESULTS
BFA Increases the Cell Surface Expression of Man-6-PIIGF 1 1 Receptors-Fibroblasts were incubated for 30 min with 5 pg/ml BFA before determining the cell surface expression of the Man-6-P/IGF I1 receptor. The binding of two high affinity ligands for the IGF I1 and Man-6-P binding sites of the receptor, '"I-IGF I1 and "'1I"an-6-P-Man4-BSA, was 1.6and 2.6-fold increased ( Fig. 1 and Table I). The binding of the monoclonal receptor antibody '2s1-2C2 was also 1.7-fold increased in BFA-treated cells. Presence of 5 pg/ml BFA during the binding at 4 "C had no effect on the binding of Man-6-P-Man4-BSA, indicating that the observed changes are not due to a direct effect of BFA on the Man-6-P/IGF I1 surface receptors. Dose-dependent binding of Man-6-P-Man4-BSA at concentrations ranging from 1 to 200 pM revealed that BFA did not change the affinity of the receptor for Man-6-P-Man4-BSA (& 13.8 PM in BFA-treated cells us. 14.2 pM in controls).
The effect of 5 pg/ml BFA reached half of its maximum within 5 min (Fig. 2 A ) . Using a 30-min pretreatment, halfmaximal stimulation was observed at 10"j M BFA (Fig. 2B). In all further experiments the cells were pretreated for 30 min " Overexpressing the human Man-6-P/IGF I1 receptor.
at 37 "C with 5 pg/ml BFA (1.8 X M). Removal of BFA from the medium showed that the effect was reversible with a tIl2 of about 30 min (Fig. 2 A ) . In presence of BFA the increased binding of '2sII"an-6-P-Man4-BSA persisted for at least 15 h. When fibroblasts were incubated for 1 and 4 h with 0.5 mM cycloheximide and then treated for 30 min with 5 pg/ml BFA and cylcoheximide, a 2.6-fold increase of cell surface Man-6-P/IGF I1 receptors was observed (not shown). The increased expression of Man-6-P/IGF I1 receptors is therefore due to redistribution of preexisting receptors and not dependent on the synthesis of new receptors.
About 20% of the Man-6-P/IGF I1 receptors at the cell surface of fibroblasts are occupied with endogenous Man-6-P-containing ligands (15). In BFA-treated cells only 9% (n = 5) of the cell surface receptors were occupied, indicating that largely unoccupied receptors are redistributed to the cell surface.
BFA   223 k 31 (5) The numbers in parentheses represent the number of independent experiments. was measured. Cells were pretreated for 30 min at 37 "C in the presence of 5 pg/ml BFA and then incubated for additional 30 min at 37 "C with "51-arylsulfatase A and BFA. The uptake of 1261-arylsulfatase A was 2.0-fold increased in BFA-treated cells compared to untreated cells (Table 11). Also the uptake of the 2C2 antibody was 2.2-fold increased (Table 11) and linear for at least 120 min in BFA-treated cells (not shown). The endocytosis of '251-Man-6-P-Man4-BSA, however, was 4.4-fold increased (Table 11). It should be noted that the binding of Man-6-P-Man4-BSA was also increased only 2fold as that of 2C2 (see Table I). In Hep G2 cells (see below) binding and uptake of Man-6-P-Man4-BSA was increased 2fold. BFA may induce conformational changes of Man-6-P/ IGF I1 receptors in BFA-treated fibroblasts which specifically favor the interaction with Man-6-P-Man4-BSA at 37 "C. This specificity may be related to the polyvalency of Man-6-P-Man,-BSA (230 mol of Man-B-P/mol BSA).
To examine whether BFA affects the transport from endosomes to lysosomes Percoll density centrifugation was performed with postnuclear supernatants obtained from cells which had internalized '"I-arylsulfatase A in the absence and presence of BFA for 15 and 45 min. In cells incubated for 15 min with '2'I-arylsulfatase A 12% of the ligand was associated with the dense lysosomal fraction. This fraction increased to 38% after an internalization period for 45 min and was not affected by BFA. When cells were incubated for up to 3 h with '251-Man-6-P-Man4-BSA, BFA increased the amount of internalized and of degraded ligands 4-fold (not shown). These results indicate that the transport of Man-6-P-containing ligands to lysosomes and the degradation therein is not affected by BFA.
Effect of BFA on the Internalization and Recycling of Man-6-PIZGF ZZ Receptor-At steady state the number of receptors that is internalized per unit of time equals that which is recycled to the cell surface. Thus, kint X [R].,, = keXt X [R]int, where kin, represents the rate of internalization, [R],,, the concentration of receptors at the cell surface, k,,, the rate of externalization, and [RIi,, the concentration of receptor available for recycling to the cell surface. An increase of the Man-6-P/IGF I1 receptor concentration at the cell surface can result from a decrease of kint, an increase of he,,, or an increase To examine whether BFA affects kint, we measured the uptake of prebound (4 " C ) ligands upon warming the cells to 16 or 20 "C. The uptake of prebound Man-6-P-Man4-BSA or 2C2 antibody by BFA-treated cells was faster and more efficient (Fig. 3B). The initial rate of internalization and the relative amount of ligand internalized during the incubation for 30-50 min were 20-40% higher than in controls. The improved internalization was accompanied by a decreased release of prebound ligands into the medium (Fig. 3C), while the rate of disappearance of ligands from the cell surface was not affected by BFA (Fig. 3A).
The redistribution of Man-6-P/IGF I1 receptors to the cell surface of BFA-treated cells must therefore result from an increase of keXt X [RIi,,, which overrides the effect of the higher kint. The externalization of internalized Man-6-P-Man4-BSA was measured to obtain information about the effect of BFA on k,,,. After binding of lZ5I-Man-6-P-Man4-BSA at 4 "C the cells were shifted to 37 "C for 3 min, and the surface bound ligands were stripped by washing with 2 mM Man-6-P. The cells containing the internalized Man-6-P-Man4-BSA were then incubated for up to 15 min at 37 "C. The incubation medium was supplemented with 2 mM Man-6-P to induce also the release of Man-6-P-Man4-BSA returning to the cell surface in a receptor-bound form and to prevent re-uptake of secreted Man-6-P-Man4-BSA. During the 15min incubation 56% of the internalized Man-6-P-Man4-BSA was released into the medium by control cells and 77% by BFA-treated cells (Fig. 4). This clearly indicates that kXt is increased in BFA-treated cells. induce the redistribution through a mechanism which is shared with or independent of the BFA-induced mechanism.
Incubation of fibroblasts for 10 min with saturating amounts (lo-' M) of IGF I and IGF I1 resulted in a 2-and 1.6-fold increase of the Man-6-P/IGF I1 receptor expression at the cell surface (26). When the cells had been treated for 30 min with BFA, IGF I and IGF I1 led to a 3.6-and 2.7-fold increase of Man-6-P/IGF I1 receptor expression (Table 111). The effects of IGF I and IGF I1 are therefore largely additive . 4. Effect of BFA on the externalization of '261-Man-6-P-Man4-BSA. Fibroblasts were incubated for 30 min with or without BFA (5 pgfml) prior to binding of lZ5I-Man-6-P-Man4-BSA for 4 h at 4 "C. After removal of unbound 1Z51-Man-6-P-Man4-BSA the cells were warmed up to 37 "C for 3 min in the absence or presence of BFA. The cells were chilled to 4 "C, and cell surface-bound lZ51-Man-6-P-Man4-BSA was displaced by Man-6-P. After washing, the cells were again warmed to 37 "C in the absence (0) or presence (0) of BFA in MEM containing 20 mM Hepes, pH 7.2, 0.1% BSA, and 2 mM Man-6-P to prevent re-uptake of ligands. At various time points the radioactivity in the medium was determined and expressed as percentage of internalized radioactivity.  to that of BFA, suggesting that BFA and the growth factors induce the receptor redistribution through independent mechanisms.

BFA Increases the Cell Surface Expression of the M, 46,000 Mannose 6-Phosphate Receptor, but Not of Transferrin Recep-
tors-The effect of BFA on two other recycling receptors in fibroblasts was examined. The surface expression of the M, 46,000 mannose 6-phosphate receptor (MPR 46) cannot be followed directly by binding of ligands since this receptor does not bind Man-6-P-containing ligands when expressed at the cell surface (11,27). Furthermore, its expression is too low to be measured by binding of receptor antibodies. We, therefore, measured the continuous uptake of the monoclonal antibody 21D3 against the MPR 46 at 37 "C under conditions analogous with that for the 2C2 antibody directed against the Man-6-P/IGF I1 receptor (see Table 11). Treatment with BFA for 30 min increased the uptake of '251-labeled 21D3 to 163 f 22% ( n = 4) of control indicating that MPR 46 expression at the cell surface is also increased by BFA.
The surface expression of transferrin receptors measured by binding of lZ5I-labeled transferrin decreased to 85 and 83% after treating cells for 0.5 and 1 h with BFA.

BFA Induces the Redistribution of Man-6-PIZGF ZI Receptors in a Variety of Cell
Types-Treatment of the human hepatoma cell line Hep G2, of Chinese hamster ovary cells, and of transfected mouse Lcells and baby hamster kidney cells, which overexpress the human Man-6-P/IGF I1 receptor for 30 min with 5 pg/ml BFA, increased the expression of Man-6-P/IGF I1 receptor 2.1-3.1-fold (Table I).
In Hep G2 cells the distribution of Man-6-P/IGF I1 receptor was also analyzed by indirect immunogold labeling. The fraction of protein A-gold particles present at the plasma membrane of BFA-treated cells was twice of that in controls. Interestingly, also the distribution of Man-6-P/IGF I1 receptors at the cell surface was affected by BFA. While in controls 8% of immunogold particles at the cell surface were associated with coated-pit areas, this fraction increased to 30% in BFAtreated Hep G2 cells (Table IV). In spite of higher number of Man-6-P/IGF I1 receptors in coated-pit areas, the uptake of Man-6-P-Man4-BSA in these cells was increased by BFA only 2-fold (not shown).

DISCUSSION
The present study describes the redistribution of Man-6-P/IGF I1 receptors induced by BFA in a variety of cell types. BFA increases the concentration of the receptor at the cell surface %fold. Formally, such an effect could result from a decrease of kint, an increase of keXt, and/or an increase of internal receptors that recycle. The increase of k,,, that was observed is likely to be compensated by the increase of kint. We assume, therefore, that the number of internal receptors  30 Percentage of gold particles at the plasma membrane. The data were calculated from 900 gold particles counted in 125 cells.
Gold particles in coated-pit areas expressed as percentage of the plasma membrane bound gold particles. In controls 110 and in BFAtreated cells 143 gold particles at the cell surface were counted.
at UNIVERSITEIT UTRECHT on September 12, 2006 www.jbc.org Downloaded from that are available for recycling to the cell surface is increased in BFA-treated cells.
Man-6-P/IGF I1 receptors can recycle from endosomes either to the cell surface or to the TGN (11). They share this dual exit from endosomes with the second Man-6-P-specific receptor, the M , 46,000 mannose 6-phosphate receptor. Interestingly, BFA induces an increase of both Man-6-P receptors at the cell surface, while the surface concentration of the transferrin receptor, which recycles largely between early endosomes and the plasma membranes (28,29) is slightly decreased in BFA-treated cells. The possibility should therefore be considered that BFA impairs the recycling of receptors to the TGN and thereby increases the number of receptors that can recycle from endosomes to the cell surface. The resialylation of Man-6-P/IGF I1 receptors in BFA-treated cells that was observed by Chege and Pfeffer (7) would imply that transport to the TGN is only impaired but not abolished. Alternatively, BFA could facilitate the recycling of receptors between the cell surface and intracellular compartments that normally are excluded from recycling.
BFA not only increases the surface expression of Man-6-P/IGF I1 receptors, but also alters the distribution at the cell surface. In Hep G2 cells the fraction of surface receptors associated with coated pits was 4-fold higher than in controls. It remains to be shown whether the coat is presented by clathrin and whether the coated pits are altered in size or number. It is conceivable that the effect of BFA on the distribution of Man-6-P/IGF I1 receptors at the cell surface is related to its stimulatory effect on the rate of internalization.
While the dramatic effect of BFA on the morphology of the Golgi and the redistribution of Golgi components to the ER are well-described (2, 6, 30), it is still unkown how BFA acts on the molecular level. The earliest effect of BFA noted so far is the rapid (within 30 s) displacement of the 110-kDa 6-COP subunit from the Golgi membranes (31). @-COP is a peripheral protein on the cytoplasmic site of the transitional ER, Golgi stack, and TGN and a major component of the nonclathrin coat of vesicles that are thought to mediate intercisternal transport in the Golgi (32,33). BFA is supposed to prevent the assembly of the nonclathrin coat required for the formation of vesicles mediating anterograde transport from the ER to the Golgi and across the Golgi (34).
The effect of BFA on the redistribution of Man-6-P receptors could result from the interaction of BFA (or its receptor) with a component required for vesicular transport within the endocytic route or the transport from endosomes to the TGN. It is also conceivable however, that the redistribution of Man-6-P receptors is secondary to the effects of BFA on the secretory route, e.g. the acceptor properties of the TGN for vesicles arriving from endosomes may be altered in BFAtreated cells. The accumulation of such vesicles may impair their formation. Notwithstanding the redistribution of Man-6-P receptors to the cell surface is a direct or an indirect effect of BFA on the endocytic route, our findings clearly demonstrate that the biological effects of BFA are not restricted to the secretory pathway.