(Received for publication, March 22, 1995; and in revised form, August 21, 1995)
From the
Efficient transferrin receptor recycling is reconstituted when
donor cytosol and ATP are added to the streptolysin O permeabilized
cells. The rate of reconstituted recycling is dependent on the
concentration of donor cytosol. The cytosol provides a factor(s)
required for the transport of transferrin from the pericentriolar
recycling compartment to the plasma membrane. N-Ethylmaleimide
treatment of permeabilized cells inhibits both the fusion of recycling
vesicles with the plasma membrane as well as the formation of
functional recycling vesicles from the pericentriolar recycling
compartment. Guanosine 5`-3-O-(thio)triphosphate (GTPS)
does not affect reconstituted recycling in the presence of an optimal
cytosol concentration. Therefore, the rate-limiting step in recycling
is not regulated by GTP-hydrolyzing proteins, and hydrolysis of GTP is
not required for endocytic recycling. GTP
S stimulates recycling
when suboptimal concentrations of cytosol are used. This stimulatory
effect is not mediated by a brefeldin A-sensitive ADP-ribosylation
factor protein. Addition of wild-type donor cytosol to permeabilized
END2 Chinese hamster ovary cells, which recycle transferrin at half the
rate of wild-type cells, reconstitutes recycling to the reduced rate of
intact END2 cells but not to the wild-type recycling rate. These
results indicate that the defect responsible for the slowed transferrin
recycling in END2 mutants is membrane associated or that the defective
protein is too large to diffuse out of the cells through the
streptolysin O pores.
Receptor-mediated endocytosis is a process used by cells for
rapid and specific uptake of extracellular macromolecules (McGraw and
Maxfield, 1991; Trowbridge et al., 1993). Most internalized
ligands are delivered to lysosomes, and most receptors are recycled
back to the cell surface to mediate further rounds of ligand
internalization. Membrane proteins are recycled to the cell surface by
a default, bulk flow process (Dunn et al., 1989; Mayor et
al., 1993). In CHO ()cells, recycling membrane is
concentrated in the pericentriolar region of the cell (Fig. 1).
This concentration of recycling membrane is a fusion-competent, stable
compartment (Yamashiro et al., 1984; Salzman and Maxfield,
1988; Marsh et al., 1995). Although the recycling compartment
is in the same general region of the cell as the trans-Golgi network
and the Golgi complex, the recycling compartment is distinct from these
organelles (Yamashiro et al., 1984, McGraw et al.,
1993). Transfer from the pericentriolar recycling compartment to the
plasma membrane is the rate-limiting step in recycling of lipids and
receptors back to the cell surface, occurring with a half-time of
12 min (Mayor et al., 1993; Presley et al.,
1993). A similar pericentriolar recycling compartment is found in other
cell lines, indicating that the pericentriolar recycling compartment is
not peculiar to CHO cells (e.g. Tooze and Huttner(1990);
Apodaca et al.(1994)). The transfer of recycling membrane from
the pericentriolar compartment to the plasma membrane is likely to
involve the formation of recycling vesicles, although there is no
direct evidence for these intermediates in recycling in CHO cells.
Figure 1: Schematic of the endocytic recycling pathway in CHO cells. Apo-Tf, iron-free transferrin; MTOC, microtubule organizing center. In CHO cells, recycling membranes (receptors and lipids) are concentrated in a fusion-competent compartment in the pericentriolar region of the cell. Transport from this compartment to the cell surface is the rate-limiting step in recycling. Recycling vesicles mediating transport between this compartment and the cell surface are shown; however, these recycling vesicles have not been identified in CHO cells.
Many vesicle-mediated membrane-trafficking steps have been reconstituted in cell-free and semi-intact cell systems (Rothman, 1994). These studies have led to the development of a model for vesicle-mediated membrane trafficking (Rothman, 1994). In this model, GTP-binding proteins and coat proteins are required for the formation of coated buds on donor membranes (Orci et al., 1986; Serafini et al., 1991). The transport vesicles are uncoated, in a process requiring GTP hydrolysis, prior to fusion with target membranes (Serafini et al., 1991). The specificity of the fusion, in part, is dependent on proteins found on transport vesicles and target membranes (v-SNAREs and t-SNAREs, respectively) (Söllner et al., 1993a; Takizawa and Malhotra, 1993). Fusion of vesicle and target membranes require the binding of NSF protein and SNAP proteins to the v- and t-SNARE complex (Söllner et al., 1993b; Südhof et al., 1993). The interactions of SNAREs may be further modulated by small GTP-hydrolyzing Rab proteins (Pfeffer, 1992; Novick and Brennwald, 1993). A number of diverse vesicle-mediated membrane transport steps (e.g. intra Golgi, endoplasmic reticulum to Golgi, TGN to plasma membrane, synaptic vesicle release, endosome-endosome fusion) require all or some of the above mentioned factors (Gravotta et al., 1990; Miller and Moore, 1991; Ostermann et al., 1993; Peter et al., 1993). As endocytic recycling is likely to be vesicle mediated, it is reasonable to suggest that some of the proteins required for these other membrane transport processes will also be required for endocytic recycling.
To determine the biochemical requirements for endocytic recycling in CHO cells, we have modified a streptolysin O toxin (SL-O) permeabilization technique previously used to examine transport from the TGN to the cell surface of CHO cells as well as other transport steps involving the fusion of vesicles with the plasma membrane (Miller and Moore, 1991; Robinson et al., 1992; Galli et al., 1994). SL-O permeabilization was chosen because it forms stable pores that are large enough to allow proteins on the order of 150 kDa to pass through the plasma membrane, and most importantly SL-O toxin can be used to selectively permeabilize the cell surface (Ahnert-Hilger et al., 1989).
To assay for endocytic recycling in SL-O toxin-permeabilized cells, the release of previously internalized transferrin (Tf) from cells is monitored. Tf is an ideal tool for monitoring endocytic recycling because it remains associated with the TR until it returns to the plasma membrane (Dautry-Varsat et al., 1983; Klausner et al., 1984). Tf is internalized through clathrin-coated pits into acidic endosomal components, where iron is released from Tf. Unlike other endocytosed ligands, apo-Tf (iron-free) is not released from its receptor in the acidic environment of endosomes. Apo-Tf remains bound to the TR, and this complex is recycled back to the cell surface. At the neutral pH of the extracellular environment, apo-Tf is released from the TR. Thus, the release of previously internalized Tf into the medium can be used to monitor the recycling of the TR back to the cell surface. The kinetics of Tf trafficking and the compartments through which it passes have been extensively documented in CHO cells (e.g. Dunn et al.(1989); McGraw and Maxfield (1990); Wilson et al.(1993)).
In this report, we demonstrate that the ability to
reconstitute efficient Tf recycling in SL-O permeabilized cells is
dependent on the addition of donor cytosol and ATP. We have conducted
studies using the alkylating agent NEM and the nonhydrolyzable GTP
analog GTPS to further explore the molecular requirements for
recycling. Furthermore, we establish the utility of this assay in
biochemical complementation studies using a CHO END2 mutant cell line.
A 24-well plate of TRVb-1 CHO cells was incubated at 37 °C, 5%
CO in MB buffer (supplemented with 3 µg of
I-Tf/ml of MB buffer) for 1 to 2 h. The incubation medium
was removed, and cells were washed once with medium 1 (150 mM NaCl, 20 mM Hepes, 1 mM CaCl
, 5
mM KCl, 1 mM MgCl
, pH 7.4). Cells were
incubated for 2 min at room temperature in a mild acid buffer (0.5 M NaCl, 50 mM MES, pH 5.0), washed three times with
ice-cold medium 1, and placed on ice. Incubation in mild acid buffer
followed by neutral washes released surface-bound Tf (Dautry-Varsat et al., 1983). The cells were washed twice with
phosphate-buffered saline (136.9 mM NaCl, 2.7 mM KCl,
8.1 mM Na
HPO
, 1.5 mM
KH
PO
, pH 6.9) at 4 °C and incubated with
200 µl/well of 0.8 units SL-O/ml for 4 min on ice. Mock-treated
cells were incubated in ice-cold medium 1. In a typical experiment,
cells in the first row of 6 wells were left intact (mock-treated), and
the other three rows were treated with SL-O. The SL-O was removed and
replaced with transport buffer (78 mM KCl, 4 mM
MgCl
, 50 mM Hepes, pH 7.2, with KOH, 2 mM DTT). The plates were transferred to a 37 °C water bath for 4
min to allow pore formation. SL-O will insert into membranes at 4
°C but will only form pores at 37 °C, so preloading the plasma
membrane with SL-O toxin at 4 °C followed by removal of unbound
SL-O prior to incubation at 37 °C ensures that the plasma membrane
is selectively permeabilized (Ahnert-Hilger et al., 1989). The
cells were checked by light microscopy for morphology characteristic of
SL-O permeabilization (e.g. Miller and Moore(1991)). In
initial experiments, uptake of the membrane-impermeant nuclear dye,
propidium iodide, was used to confirm permeabilization. Estimations
using this method to monitor permeabilization showed that 95% (or
greater) of the cells were permeabilized by the SL-O treatment.
Following the incubation at 37 °C, cells were placed on ice and
incubated in fresh ice-cold transport buffer for 10-30 min. The
transport buffer was removed, and 300 µl of the regeneration
solutions were added to the respective wells: donor cytosol and ATP
regeneration solution (1 mM ATP, 8 mM creatine
phosphate, and 40 units/ml creatine phosphokinase) in transport buffer,
an ATP regeneration solution in transport buffer, or transport buffer
alone. Intact cells were incubated in medium 1. All regeneration
solutions contained approximately 20 µg/ml of unlabeled
Fe
Tf. For most experiments, mouse liver cytosol at 3 mg/ml
was used. Cells were incubated in the regenerating solutions for 5 min
at 4 °C, and the plate was then transferred to a 37 °C water
bath. The medium (effluxed Tf) was removed and collected at various
time points (one well per time point, six time points per condition),
and the cells were washed once in phosphate-buffered saline. This wash
was pooled with the efflux medium. The cells were solubilized in 1%
Triton X-100, 0.1 N NaOH. The efflux and cell-associated cpm
were determined, and the rate of efflux was calculated as the slope of
the line of a plot of the natural log of the fraction Tf
cell-associated versus time (Johnson et al., 1993).
It is not practical to correct each value for nonspecific radioactivity, as is commonly done for studies of intact cells. The data presented in this paper are derived from total radioactivity per well. However, in other experiments of intact cells, using the same cell line and the same batches of iodinated Tf as used for the studies presented in this report, nonspecific radioactivity (that is, not competed by incubation with a 200-fold excess of unlabeled Tf) did not account for more than 10% of the total cell-associated radioactivity. Initial experiments revealed that nonspecific counts in the SL-O permeabilized cell assay fell within 10% of total counts.
Figure 2: Tf recycling in CHO cells. The efflux (open circles) and cell-associated (closed circles) cpm of iodinated Tf per well from a representative experiment are shown. In panel A, the data from mock SL-O treated cells are shown (intact). In panels B through D, the data shown are from SL-O permeabilized cells incubated in 3 mg/ml mouse liver cytosol and ATP-regenerating system in transport buffer (B), an ATP-regenerating system in transport buffer (C), or transport buffer alone (D). The total cpm per time point (efflux plus cell associated) did not vary more than 18% within a given condition.
In this procedure, the
SL-O toxin was bound to the cells at 4 °C, and excess SL-O was
removed before warming the cells to 37 °C. The technique ensures
that the plasma membrane is selectively permeabilized and that
intracellular organelles are left intact (Ahnert-Hilger, et
al., 1989). To confirm the integrity of internal membranes, SL-O
permeabilized cells incubated with cytosol, ATP, and an ATP
regenerating system were stained using wheat germ agglutanin (WGA) (Fig. 3). WGA binds with high affinity to high molecular weight
acidic glycoproteins found primarily on the plasma membrane, in the
lumen of endosomal compartments and Golgi, and to some extent in the
nuclear envelope (Fitzgerald et al., 1981; Raub et
al., 1990). To determine accessibility of internal membrane
compartments to rhodamine-WGA (Rh-WGA), two different experiments were
performed. In one, following a 15-min, 37 °C incubation with
cytosol and ATP, SL-O permeabilized cells were incubated with 1.0
µg/ml Rh-WGA on ice for 30 min (Fig. 3B). In the
other, 1.0 µg/ml Rh-WGA was added to the cytosol during the 15-min
incubation at 37 °C (Fig. 3C). In a control sample,
SL-O permeabilized cells following a 15-min incubation at 37 °C
with cytosol and ATP were fixed and permeabilized with detergent
(saponin, 100 µg/ml) and then incubated with Rh-WGA. In
detergent-permeabilized cells, WGA stained the plasma membrane and
intracellular membrane compartments (Fig. 3A). In SL-O
permeabilized cells not treated with detergent, WGA stained the plasma
membrane and the nuclear envelope; however, it does not stain
intracellular membrane compartments (Fig. 3, B and C). This result demonstrates that the SL-O permeabilization
procedure does not permeabilize internal membrane compartments.
Reconstituted Tf recycling is also blocked by incubation at 4 °C,
regardless of the addition of cytosol or ATP (not shown). This result
is consistent with the 4 °C block of Tf recycling in intact cells.
To demonstrate that the Tf released in the medium is not attached to
membrane sheets, the medium was collected following a 15-min efflux and
was spun for 1 h at 100,000 g. Approximately 90% of
the counts released from intact cells were in the supernatant, and
approximately 87% of the counts released from SL-O permeabilized cells,
incubated in cytosol and ATP, were in the supernatant. Furthermore, the
Tf released from SL-O permeabilized cells can be precipitated with
anti-Tf antibody (not shown). In aggregate, these results demonstrated
that the radioactivity in the medium was Tf released from cells by
fusion of recycling vesicles with the plasma membrane, since if intact
vesicles containing Tf were released by lysis of cells, the Tf in the
lumen of the vesicles, would not be accessible to the antibody.
Figure 3: Integrity of internal membranes is maintained in SL-O permeabilized cells. In panels A through C, cells were permeabilized with SL-O and incubated in cytosol, ATP, and an ATP regenerating system followed by 15 min at 37 °C to allow recycling. In panel A, the cells were fixed and treated with Rh-WGA in the presence of detergent (100 µg/ml saponin) for 30 min. In panel B, the cells were returned to 4 °C, incubated in Rh-WGA in transport buffer for 30 min, washed for 10 min in transport buffer to remove excess label, and fixed. In panel C, Rh-WGA was added to the cytosol solution during the 37 °C incubation. At the end of the assay, the cells were returned to 4 °C, washed for 10 min in transport buffer, and fixed. Panel D is a schematic of the optical section photographed in these experiments. The plasma membrane appears as a ring at the optical section (solid arrows). The edge of the cell at its bottom was only visible if planes below the optical section contained fluorescently labeled structures. In panel A, the outer edge of the cell bottom is apparent (open arrow) because internal structures are labeled. In panels B and C, the outer edge of the cell (open arrows) bottom is not as apparent because internal structures are not labeled.
The
loss of cell-associated Tf from CHO cells fits a single exponential
decay. The recycling rate constant is the slope of a plot of the
natural log of the fraction of Tf cell-associated versus time.
In all permeabilized cell conditions, the fraction of Tf that is
cell-associated at time zero is similar to that found in intact cells,
demonstrating that SL-O permeabilization does not result in a
significant aberrant loss of Tf from cells, as would be expected if the
permeabilization was destroying the integrity of the recycling
compartment (Fig. 4A). The data of Fig. 2plotted to calculate the recycling rate constant are shown
in Fig. 4A. In this experiment, the rate of Tf efflux
in the presence of cytosol is 70% of the intact rate, and that of
incubation with ATP or transport buffer alone is 34 or 21% of the
intact rate, respectively. A summary of a number of experiments is
shown in Fig. 4B. SL-O permeabilized cells recycle Tf
at 80% of the intact rate when incubated with 3 mg/ml donor
cytosol and ATP,
35% when incubated with ATP, and
25% when
incubated with transport buffer alone. Both the initial rate of Tf
recycling and the extent of Tf released from the cells is dependent on
addition of donor cytosol and ATP to the permeabilized cells (Fig. 4C). After 20 min of incubation, little further
Tf is released from either permeabilized or intact cells. In both
intact cells and permeabilized cells incubated with donor cytosol,
about 20% of the total Tf remains internal after a 60-min incubation at
37 °C. An explanation for this plateau at 20% is that the data are
not corrected for nonspecific Tf binding, which is typically 10% of the
total cpm bound (see ``Experimental Procedures''). Regardless
of the reason for the plateau at 20%, Tf recycling from permeabilized
cells incubated with ATP and donor cytosol is similar to recycling in
intact cells.
Figure 4:
Determination of Tf recycling rate
constant in intact and SL-O permeabilized CHO cells. In panel
A, the rate lines for a representative experiment (same data as Fig. 2) are shown. The natural log of the percent
cell-associated cpm is plotted, and a best fit line is drawn. Open
circles are values for intact cells. Filled circles are
values for permeabilized cells incubated in 3 mg/ml mouse liver donor
cytosol and an ATP-regenerating system. Open squares are
values for permeabilized cells incubated in an ATP regenerating system
and no donor cytosol. Filled squares are values for
permeabilized cells incubated in transport buffer alone. In panel
B, the average values from a number of experiments are presented.
Each time point is the mean of at least 14 experimental measurements
± S.E. Symbols in panel B are the same is in panel A. In panel C, the results of a Tf recycling
experiment extended out to 60 min is shown. The initial rates (first 20
min) are comparable to rates obtained in other experiments (e.g.
panel A). Symbols are the same as in panel A.
The open triangles are values for cells incubated with 6 mg/ml
donor mouse liver cytosol. Panel C, n
2.
Figure 5:
Reconstituted Tf recycling is dependent on
the concentration of donor cytosol. The average recycling rate
constants as a function of donor cytosol are presented. The data
presented represent experiments performed with mouse liver cytosol.
Plus (+) ATP denotes incubation with 1 mM ATP and an
ATP-regenerating system (see ``Experimental Procedures'').
The data for the bar labeled ``boiled'' were
collected using cytosol that was boiled for 5 min prior to use in the
assay. The data for the bar labeled ``gel filtered''
were collected using cytosol that had been passed over a gel filtration
column (PD-10, Pharmacia) to exclude molecules less than 5000 Da.
Bovine serum albumin was added and used at a concentration 3 mg/ml in
place of donor cytosol. Error bars represent S.E. (n 2).
The efflux of fluorescent Tf during the basic incubation conditions is shown in Fig. 6. After a 20-min incubation, most of the Tf has been released from intact cells, with the residual fluorescence visible in the pericentriolar region (Fig. 6, A and B). SL-O permeabilization does not alter the morphology of the Tf-containing pericentriolar recycling compartment (Fig. 6C). Consistent with the biochemical studies, most of the internal Tf is released from SL-O permeabilized cells during a 20-min incubation with donor cytosol and ATP (Fig. 6, C and D). Unlike intact cells, the residual Tf fluorescence in SL-O permeabilized cells incubated with cytosol and ATP is not solely concentrated in the pericentriolar region but is also found in a punctate pattern distributed throughout the cell (Fig. 6, B and D). In SL-O permeabilized cells incubated for 20 min with ATP alone or in transport buffer alone, fluorescent Tf remains in the pericentriolar region (Fig. 6, E and F). These results demonstrate that a factor provided by the donor cytosol is required for transport of Tf from the pericentriolar recycling compartment to the plasma membrane.
Figure 6: Fluorescent Tf recycling assayed using fluorescent microscopy. Intact CHO cells were loaded to steady state with fluorescein Tf. The cells were then treated as described under ``Experimental Procedures.'' After 20 min of efflux (or before efflux for 0-min panels), cells were fixed in 3.7% formaldehyde. Panel A shows intact cells fixed before efflux (0 min). Panel B shows intact cells fixed after 20 min of efflux. The arrows in panels A, B, C, E, and F indicate Tf in the pericentriolar recycling compartment. The open arrows in panel D indicate residual Tf not in the recycling compartment. Panel C is SL-O permeabilized cells at 0 min. Panel D is permeabilized cells incubated in cytosol (3 mg/ml), ATP, and an ATP-regenerating system for 20 min at 37 °C before fixation. Panel E is the morphology of permeabilized cells incubated in ATP and an ATP-regenerating system for 20 min at 37 °C before fixation. Panel F is permeabilized cells incubated in transport buffer alone for 20 min at 37 °C before fixation.
Figure 7: Tf recycling in END2 cells supported by mouse liver cytosol. The END2 cells were treated identically to wild-type cells. The first bar represents the rate of Tf recycling in intact END2 cells. The second and third bars represent the rate of Tf recycling in SL-O permeabilized END2 cells incubated in either 6.0 or 3.0 mg/ml cytosol and the ATP-regenerating system. The fourth bar is Tf recycling in SL-O permeabilized END2 cells incubated in the ATP and ATP-regenerating system only. Error bars represent S.E. (n = 3). In bar one and four (intact and ATP only treated), S.E. = ±0.0003. The absolute rates of Tf recycling vary with preparations of cytosol. Wild-type cells in matched experiments were two times the rate of intact END2 cells.
Figure 8:
Tf
recycling in permeabilized CHO cells in which donor cytosol or
permeabilized cells were treated with NEM. The average values of four
experiments (±S.E.) to determine the recycling rate constants
are shown. All rate lines obtained for NEM experiments resulted in
linear lines (not shown). The data were then analyzed to determine the
percent inhibition. Percent inhibition of Tf recycling supported by
cytosol and ATP was calculated as described previously (Beckers et
al., 1989). In this analysis, 100% inhibition represents the rate
supported by addition of ATP alone, and 0% inhibition represents the
rate supported by the addition of 3 mg/ml donor cytosol and ATP (i.e. complete conditions). Thus, percent inhibition
represents inhibition of Tf recycling promoted by addition of donor
cytosol. In panel A, the results of NEM treatment of donor
cytosol are presented. The results presented in the bar labeled pretreatment at 37 °C with no ATP were collected from
reactions in which the cytosol was preincubated at 37 °C in the
absence of ATP for 30 min at 37 °C. This treatment inactivates NSF
(Block et al., 1988). This cytosol was supplemented with ATP
and an ATP regenerating system when added to the permeabilized cells. Panel B represents the results of experiments where the
permeabilized cells were treated with NEM for 15 min at 4 °C. The
NEM was quenched by incubation for 15 min at 4 °C in transport
buffer supplemented with DTT (at 2 the concentration of NEM).
The percent inhibition of recycling is shown for cells treated with 1
or 2 mM NEM and cells pretreated with NEM at 37 °C for 4
min followed by 15 min at 4 °C in transport buffer containing
DTT.
SL-O
permeabilized cells were treated with NEM, and untreated donor cytosol
was added to determine if an NEM-sensitive factor(s) required for Tf
recycling remained associated with the permeabilized cells. NEM
treatment of permeabilized cells significantly inhibited
cytosol-dependent recycling, reducing the rate by 70% (Fig. 8B). This demonstrates a requirement for a
NEM-sensitive factor that remains associated with cells following SL-O
permeabilization. Complete inhibition of cytosol-supported Tf recycling
is achieved by pretreating the SL-O permeabilized cells for 4 min at 37
°C with 2 mM NEM, whereas identical treatment of donor
cytosol only partially inhibits its ability to support recycling (Fig. 8B). These results demonstrate that Tf recycling
requires a cell-associated NEM-sensitive factor, and although they do
not demonstrate a role for NSF, they are consistent with a requirement
for NSF in Tf recycling.
Figure 9:
CHO
cells were labeled to steady state with rhodamine-Tf. SL-O
permeabilized and intact (panels A and B,
respectively) cells were treated with 1 mM NEM for 15 min at 4
°C followed by 15 min in 2 mM DTT at 4 °C, or SL-O
permeabilized and intact cells were incubated in transport buffer for
30 min at 4 °C (panels C and D, respectively).
Intact cells (B and D) were then incubated in
transport buffer with FeTf and SL-O permeabilized cells (A and C) were incubated in 3 mg/ml cytosol and ATP
for 5 min at 4 °C. The cells were warmed to 37 °C 20 min as in
the normal recycling assay. The small arrows in panels A and B indicate Tf containing vesicles near the plasma
membrane, and the large arrow indicates Tf in the
pericentriolar recycling compartment.
Figure 10:
A, treatment of SL-O permeabilized CHO
cells with GTPS and BFA. GTP
S (50 µM) was added
to the regenerating solutions (cytosol and ATP or ATP only) and was
present throughout the duration of the assay. The experiments represent
the average of at least four individual experiments (n
4
± S.E.). The values compared percent stimulation calculated from
the rates of matched experiments. Percent stimulation is calculated as
the rate of efflux in the presence of GTP
S divided by the rate of
efflux of the same condition (concentration of cytosol) in the absence
of GTP
S. Additionally displayed in this panel is the pretreatment
with BFA. 5 µg/ml BFA was added to the
I-Tf
incubation media (see ``Experimental Procedures'') 15 min
prior to the start of the assay. BFA was maintained at 5 µg/ml
throughout the remainder of the assay to prevent recovery from any
effects of BFA. BFA treatment did not exceed 2 h total (time at 37 and
4 °C) and did not exceed 1 h at 37 °C. The last bar of
the graph represents experiments conducted after first incubating the
cells in 10 µg/ml nocodazole. The nocodazole was added to the
I-Tf incubation media for 1 h and was maintained
throughout the assay. For all experiments where GTP
S was added to
cytosol, the cytosol was first passed over a gel filtration column to
remove excess free nucleotides. B, competition curve of GTP to
50 µM GTP
S. Increasing concentrations of GTP was
added to an ATP-regenerating system, and 50 µM GTP
S,
which was then added to cytosol-depleted SL-O permeabilized cells. The
rate of efflux in experimental conditions is compared to cells that
were treated with only an ATP regenerating system. Percent stimulation
was calculated as in A. Values represent the average of n
2 ± S.E.
GTPS
stimulated Tf recycling in SL-O permeabilized cells when added to
reactions containing suboptimal concentrations of donor cytosol (Fig. 10A). Tf recycling was stimulated by
60%
when 50 µM GTP
S was added to permeabilized cells in
the absence of donor cytosol. The stimulatory effect of GTP
S
decreased with increasing amounts of cytosol. Stimulation of recycling
was observed over a range of GTP
S concentrations, from 50 to 200
µM GTP
S (not shown).
The stimulation of Tf
recycling by GTPS when added to the reconstitution system in the
absence of donor cytosol is blocked by addition of an excess of GTP. A
5-fold excess of GTP only partially reduces the stimulation observed in
the absence of added GTP, whereas a 10-fold excess of GTP is required
to significantly diminish the GTP
S stimulation of recycling (Fig. 10B).
GTPS stimulates endosome-endosome
fusion in suboptimal cytosol concentrations (Mayorga et al.,
1989). It is proposed that GTP
S promotes the association of a
member of the ADP-ribosylation factor family (ARF) with endosome
membranes and that the membrane-bound ARF recruits other soluble
proteins required for fusion (Lenhard et al., 1992; Colombo et al., 1992). GTP
S stimulation of recycling could occur
by a similar mechanism. The fungal metabolite, brefeldin A, blocks
exchange of GTP for GDP on ARF and consequently inhibits association of
ARF to membranes (Donaldson et al., 1992; Helms and Rothman,
1992; Dascher and Balch, 1994). If the effect of GTP
S on recycling
in suboptimal donor cytosol concentrations is mediated by an ARF
protein, then preincubation of permeabilized cells with brefeldin A
would block GTP
S stimulation of recycling. Cells were preincubated
with radioactive Tf for 1.5 h with 5 µg/ml brefeldin A included
during the last 15 min of this incubation and in all subsequent
incubations. The cells were permeabilized and the effect of GTP
S
on recycling determined. Brefeldin A did not affect GTP
S
stimulation of recycling (Fig. 10A), although it did
cause tubulation of the Tf-containing recycling compartment in CHO
cells (McGraw et al., 1993). These results suggest that
GTP
S stimulation of Tf recycling in suboptimal cytosol
concentrations is not mediated by a brefeldin A-sensitive member of the
ARF family.
To determine whether the GTPS stimulation of
recycling in suboptimal cytosol concentrations is dependent on the
microtubule cytoskeleton, microtubules were depolymerized with
nocodazole, and recycling in permeabilized cells was measured.
Depolymerization of microtubules did not affect the cytosol-dependent
reconstituted recycling (not shown) nor did it affect the GTP
S
stimulation of recycling in suboptimal cytosol concentrations (Fig. 10A). These results are in agreement with studies
of intact CHO cells, where it has been shown that recycling is not
dependent on an intact microtubule cytoskeleton (McGraw et
al., 1993).
In this report, we demonstrate that efficient Tf recycling from SL-O permeabilized CHO cells is reconstituted when the cells are incubated with donor cytosol and ATP. The rate of reconstituted recycling is dependent on the concentration of cytosol added to the permeabilized cells. Fluorescent microscopy analysis of reconstituted recycling demonstrates that donor cytosol provides an activity required for efficient transport of Tf from the pericentriolar recycling compartment. Addition of ATP to permeabilized cells in the absence of donor cytosol promotes only a modest increase in recycling over that achieved by incubation in transport buffer alone. The results of this condition (ATP only) serve as an internal control for the degree and extent of SL-O permeabilization in this system. The reconstitution of Tf recycling in SL-O permeabilized CHO cells supplemented with only ATP was previously reported (Galli et al., 1994). In that study, the requirement for cytosol in reconstitution of recycling was not investigated. Our observation that efficient recycling is dependent on donor cytosol is in agreement with results of a study of Tf recycling to the basolateral membrane in semi-intact Madin-Darby kidney cells (Podbilewicz and Mellman, 1990).
The slowed Tf recycling characteristic of CHO mutants of the END2 complementation group can be recapitulated in permeabilized END2 cells. This result provides evidence that the system is faithfully reconstituting Tf recycling. As in wild-type cells, cytosol stimulates reconstituted recycling in END2 cells above the rate supported by ATP alone. Donor cytosol from mouse liver or wild-type CHO cells restores Tf recycling to the slow rate characteristic of intact END2 cells. These ``wild-type'' donor cytosols, however, are unable to restore wild-type rates of recycling to permeabilized END2 cells. These results indicate that the factor responsible for the reduced rate of Tf recycling in mutants of the END2 complementation group is membrane associated or is too large to diffuse through the SL-O pores.
Tf recycling is inhibited by mild NEM treatment of permeabilized cells. Finding Tf in a punctate distribution near the plasma membrane following mild NEM treatment of permeabilized cells is consistent with a block in fusion of recycling vesicles with the membrane and consequently is consistent with NSF being at least one of the factors inactivated by the mild treatment of cells with NEM. Tf recycling is partially inhibited when the v-SNARE, cellubrevin, is cleaved by tetanus toxin, which is consistent with a role for NSF in Tf recycling (Galli et al., 1994). The failure of untreated donor cytosol to rescue the inhibition in recycling caused by NEM treatment of permeabilized cells may be because the NSF trimer is too large to efficiently diffuse through the SL-O pores, either in or out of the permeabilized cells, or that the NSF required for recycling may be associated with the membrane. In this regard, it has been suggested that NSF is required for vesicle formation (Wattenberg et al., 1992). Although our findings are consistent with a requirement for NSF in recycling, we cannot rule out the possibility that NSF is not required and the mild NEM treatment is inactivating other proteins required for recycling. For example, endosome-TGN transport involves a protein other than NSF that is sensitive to NEM (Goda and Pfeffer, 1991). In this regard, it is interesting that mild NEM treatment inhibits transport of Tf from the pericentriolar recycling compartment. This block could be due to NEM inactivation of a protein other than NSF. Rigorous demonstration that NSF is necessary for Tf recycling requires the use of reagents that specifically inactivate NSF (e.g. antibodies). The recent finding that apical transport in Madin-Darby kidney cells does not require NSF provides precedent for NSF-independent fusion with the plasma membrane (Ikonen, et al., 1995).
Reconstituted Tf recycling is not
inhibited by GTPS, and therefore, hydrolysis of GTP is not
required for Tf recycling. The failure of GTP
S to inhibit
recycling distinguishes the mechanism for recycling from the mechanism
for transport from the TGN to the cell surface because the latter is
inhibited by GTP
S (Miller and Moore, 1991). In light of the
findings that G proteins of the Rab and ARF families and heterotrimeric
G proteins are involved in intracellular trafficking of membrane
proteins, it is surprising that GTP
S does not affect reconstituted
recycling supported by donor cytosol (van der Slujis et al.,
1992; D'Souza-Schorey et al., 1995; Bomsel and Mostov,
1992). The recycling assay described in this report measures transport
from the pericentriolar recycling compartment to the plasma membrane (Fig. 1). It may be that GTP hydrolysis is required in a earlier
step in the endocytic pathway, such as transport from sorting endosomes
to the recycling compartment. GTP
S inhibition of trafficking steps
preceding transport from the pericentriolar recycling compartment would
not be detected in this assay because at steady-state there is
approximately five times as much Tf in the pericentriolar recycling
compartment as in sorting endosomes (Mayor et al., 1993).
Additionally, this assay may not be sensitive to GTP
S effects on
components required to support multiple rounds of recycling vesicle
formation and consumption. Regardless, it is interesting that the
overall rate-limiting step in the constitutive recycling of membrane
back to the cell surface does not require GTP hydrolysis. Future
studies using the reconstituted recycling assay will provide insight
into the molecular mechanism of regulating recycling.
Stimulation of
fusion with the plasma membrane is commonly observed for regulated
secretion events (Gomperts, 1990). GTPS stimulates Tf recycling in
suboptimal cytosol concentrations. GTP
S may promote the
association of soluble proteins required for recycling to the recycling
compartment, and hence, in limiting amounts of added donor cytosol,
GTP
S would increase recycling. Although the permeabilized cells
are preincubated to allow soluble proteins to diffuse from cells, some
fraction of the cytosolic proteins will remain inside the cells.
Finding GTP
S has its greatest effect on recycling in the absence
of donor cytosol is consistent with the interpretation that GTP
S
is promoting the association of soluble proteins with the recycling
compartment membrane. Our results are similar to those described for
endosome-endosome fusion (Mayorga et al., 1989). GTP
S
stimulates endosome-endosome fusion at suboptimal cytosol
concentrations in a cell-free assay (Mayorga et al., 1989). It
is proposed that GTP
S promotes the recruitment of ARF and other
soluble proteins to endosome membranes (Lenhard et al., 1992;
Colombo et al., 1992). ARF proteins are required for the
binding of coat proteins to membranes (Donaldson et al., 1992;
Helms and Rothman, 1992; Stamnes and Rothman, 1993; Robinson and Kreis,
1992) and have been implicated in a number of membrane transport
processes (Balch et al., 1992; Lenhard et al., 1992).
Brefeldin A blocks exchange of GTP for GDP on ARF and blocks ARF
association with membranes (Donaldson et al., 1991; Helms and
Rothman, 1992). GTP
S stimulation of recycling in suboptimal
concentrations of donor cytosol is not affected by brefeldin A,
suggesting that the stimulatory effect of GTP
S is not due to an
increase in an ARF association with recycling membrane. In future
studies, the reconstitution system will be used to explore the role of
G proteins in regulating endocytic recycling.
A trivial explanation
for the failure of GTPS to effect recycling in the presence of
cytosol is that free nucleotides added with the donor cytosol block the
effect of GTP
S. This is extremely unlikely. First, in other
studies the effects of GTP
S have been seen using similar gel
filtration techniques for removing free nucleotides from the cytosol
(Melancon et al., 1987; Mayorga et al., 1989; Carter et al., 1993) Second, we have demonstrated that a 10-fold
excess of GTP is required to compete with the GTP
S. Thus, the
gel-filtered or dialyzed cytosol would have to provide approximately 2
mM GTP to effectively compete 200 µM GTP
S.
It is unlikely that the concentrations of free nucleotides are this
high in the gel-filtered or dialyzed cytosol.
Reconstituted Tf
recycling shares some of the characteristics of other
membrane-trafficking processes: inhibition by NEM, dependence on ATP
and donor cytosol, and partial inhibition by tetanus toxin cleavage of
cellubrevin (Galli et al., 1994). Unlike many of these other
processes, cytosol-dependent Tf recycling is not inhibited by
GTPS. This finding may point to important mechanistic differences
among various membrane transport steps. In this regard, it is of
interest to note that the recent findings that demonstrate apical and
basolateral transport in Madin-Darby kidney cells have different
molecular requirements (Ikonen et al., 1995)