Steady-State Localization of GPI-GFP and CD59 in PM and Golgi Membranes
GPI-GFP behaved as a lipid raft–associated protein, being incorporated into detergent-resistant domains. When cells were extracted with 1% Triton X-100 before fixation, GPI-GFP was resistant to this treatment, whereas vesicular stomatitis virus G protein was efficiently removed ( A; Mayor and Maxfield 1995
). The steady-state distribution of GPI-GFP in COS-7, NRK, HeLa, and MDCK cells included two major pools, one at the cell surface and one in a juxtanuclear compartment ( B). This compartment (see boxed area in B) was identified as the Golgi complex rather than recycling endosomes (which frequently have a similar distribution) by colocalization with a Golgi resident enzyme, mannosidase II, and lack of colocalization with endocytosed transferrin ( and ). Typically, 90% of the total cellular GPI-GFP fluorescence appeared on the cell surface, whereas ~10% was found in the Golgi complex.
Figure 1 Localization of specific GPI-anchored proteins to the Golgi complex. (A) Triton X-100 extraction of COS-7 cells expressing GPI-CFP (green) and VSVG-YFP (red). (B) GFP fluorescence from GPI-GFP expressed in COS-7 cells. Similar distributions were seen (more ...)
We next asked whether there are endogenous GPI-anchored proteins that share the same distribution as GPI-GFP. Antibody staining of HeLa cells expressing GPI-GFP showed that CD59, a ubiquitous GPI-anchored protein (Davies and Lachmann 1993
), is also found both in a juxtanuclear, intracellular pool and on the cell surface, even after prolonged cycloheximide treatment to chase nascent protein out of the secretory pathway ( E). The juxtanuclear pool colocalized extensively with GPI-GFP and was further identified as the Golgi complex by labeling with antibodies against the Golgi protein GM130. A chimera with GFP fused to the NH2
terminus of CD59 (CD59-GFP) expressed in NRK cells also had a distribution similar to that of GPI-GFP and endogenous CD59. This included a juxtanuclear pool that colocalized with mannosidase II after cycloheximide treatment, but not with transferrin-containing recycling endosomes ( F, data not shown). In contrast, antibodies against another GPI-anchored protein, the folate receptor, stained structures which were separate from the Golgi complex, many of which could be labeled with endocytosed transferrin ( G; Mayor et al. 1998
). These observations indicate that GPI-GFP, CD59, and a GFP fusion based on CD59 are found in both the PM and the Golgi complex, whereas a third GPI-anchored protein, the folate receptor, has a different distribution that overlaps with transferrin-containing endosomes.
Constitutive Cycling of GPI-GFP between the Golgi Complex and Cell Surface
As a direct test of whether the Golgi pool of GPI-GFP is dependent on a flux of newly synthesized protein from the ER, GPI-GFP–expressing cells were incubated in cycloheximide (which blocks protein synthesis) for 5 h, and the ratio between the mean fluorescence intensity of the Golgi region and that of the rest of the cell was calculated at different time points ( C). The relative Golgi fluorescence of GPI-GFP declined slightly during the first 2 h of the experiment, but then remained constant. This suggests that only a small proportion (15–25%) of Golgi fluorescence is derived from newly synthesized GPI-GFP, with the remainder the result of active maintenance of GPI-GFP in the Golgi complex.
Selective photobleaching provided a direct way of addressing whether GPI-GFP in the Golgi complex exchanges with GPI-GFP on the PM. The Golgi pool of GPI-GFP was selectively photobleached in cells where protein synthesis had been inhibited, and recovery of fluorescence into the Golgi region was followed over time ( A and Video 1). After bleaching the Golgi region, there was no increase in total cellular fluorescence (since protein synthesis was inhibited). Nevertheless, Golgi fluorescence rapidly recovered as cell surface fluorescence decreased. The ratio between mean fluorescence intensity in the Golgi region and that outside of this region returned to the same value as was observed before the bleach, implying that all of the GPI-GFP in the Golgi region is constantly exchanging with the non-Golgi pool (i.e., the PM). Photobleaching of the Golgi pool of CD59-GFP under the same conditions gave similar results ( B), and, as described above, the Golgi pool of endogenous CD59 was not chased out by addition of cycloheximide. This implies that the Golgi pools of endogenous CD59, CD59-GFP, and GPI-GFP are maintained by constant exchange with the PM, rather than by new protein synthesis.
Kinetic analysis of quantitative data obtained from the photobleaching experiments permitted an estimation of the rate constants characterizing transport of GPI-GFP between PM and Golgi complex ( D). Standard methods of kinetic analysis were used, with the PM and Golgi pools of GPI-GFP treated as two compartments exchanging via first order processes, each with a characteristic rate constant (see Materials and Methods). Least squares fitting of these data yielded 0.111 min−1 as the rate constant governing GPI-GFP transport from Golgi to PM, and 0.0042 min−1 for PM to Golgi transport. Given these rate constants, an average GPI-GFP molecule resides in the PM for ~200 min and in the Golgi complex for ~9 min (mean residence time in a given compartment being the reciprocal of the rate constant for exit from that compartment).
Repetitive photobleaching of a small area of the PM eliminated GPI-GFP fluorescence from the entire PM pool and gradually depleted the Golgi pool to 10–20% of its initial fluorescence intensity ( E and Video 2), as expected if GPI-GFP fluorescence in the Golgi complex is dependent on constant supply from the PM. The cells in this experiment were not treated with cycloheximide, so the data again imply that the majority of the steady-state pool of GPI-GFP in the Golgi complex is dependent on constant exchange with the PM.
We used an independent approach to confirm that GPI-GFP returns to the Golgi after exposure at the cell surface. GPI-GFP with an additional myc epitope was expressed in COS-7 cells. Anti-myc antibody was taken up to the Golgi apparatus in transfected cells, whereas no internalization of antibody was detected in neighboring nontransfected cells ( F).
Exocytic Transport Intermediates Containing GPI-GFP
Continuous cycling of GPI-GFP between the Golgi and the cell surface implies that GPI-GFP should be present in both exocytic and endocytic transport intermediates. To resolve exocytic intermediates carrying GPI-GFP out from the Golgi, the non-Golgi pool of GPI-GFP was eliminated by photobleach. Subsequent time-lapse imaging revealed numerous transport intermediates containing GPI-GFP rapidly emerging from the Golgi complex ( and Video 3, A and B). These transport intermediates appeared as small spherical or tubular structures and they moved along curvilinear tracks (Video 3, A and B). No motile transport intermediates were seen when microtubules were depolymerized with nocodazole treatment (data not shown). Intermediates were still observed in cycloheximide-treated cells. The PM fluorescence gradually recovered after the photobleach (), so the intermediates appear to function in delivery of GPI-GFP to the cell surface.
GPI-GFP Is Found in Endocytic Structures That Contain STxB and CTxB, but Not Transferrin
We examined narrow optical sections of fixed cells that had been labeled with several endocytic markers. A high proportion of those GPI-GFP–positive structures found scattered throughout the cytoplasm were labeled with 10K dextran after a 15 min uptake (36% of pixels in GPI-GFP–positive structures were also positive for dextran; see Materials and Methods; A). Those GPI-GFP structures that did not contain dextran could well represent separate exocytic intermediates, as visualized in . GPI-GFP–positive structures did not overlap with endocytosed transferrin after continuous uptake for 20 min (2.5% of pixels in GPI-GFP structures contained transferrin; B) or after shorter periods of uptake (data not shown). Furthermore, GPI-GFP–containing structures did not colocalize with the early endosomal marker EEA1 (3% of pixels in GPI-GFP structures contained EEA1; C; Stenmark et al. 1996
Figure 4 Characterization of endosomes containing GPI-GFP, STxB, and CTxB. In all images the dashed boxes indicate the area shown in the adjacent color panel. Color images have all been processed by adjusting the black level and maximal pixel intensity level in (more ...)
We next ascertained the distribution of STxB and CTxB, which, like GPI-GFP, are associated with lipid rafts and traffic from the cell surface to the Golgi complex. The intracellular distribution of structures containing CTxB after 30 min of continuous uptake from the PM was very similar to that of STxB, although PM labeling was stronger for CTxB (87% of pixels in CTxB structures contained STxB; D), arguing that STxB and CTxB share common endocytic trafficking pathways. In GPI-GFP–expressing cells, many of the endocytic structures containing CTxB or STxB also contained GPI-GFP (42% of pixels in CTxB structures contained GPI-GFP, 71% of pixels in GPI-GFP structures contained CTxB; E, not shown in the case of STxB). Those that did not contain GPI-GFP are likely to represent transferrin-containing endosomes, since endocytosed STxB and CTxB also partially colocalized with transferrin-labeled early and recycling endosomes (30% of pixels in CTxB structures contained Tf; F). The presence of STxB in these endosomal compartments has been reported previously (Mallard et al. 1998
). Thus, GPI-GFP accumulates to detectable levels in a population of endocytic structures which contain STxB and CTxB, but not transferrin receptor or EEA1, whereas STxB and CTxB are found both in GPI-GFP–labeled endocytic structures and those which contain transferrin.
GPI-GFP, CTxB, and STxB Enter the Golgi Complex in the Same Transport Intermediates
We used selective photobleaching and time-lapse imaging to visualize delivery of GPI-GFP to the Golgi complex. A series of images of a small region of the cell adjacent to the Golgi complex were rapidly collected immediately after photobleaching of the GPI-GFP fluorescence associated with the Golgi complex and the surrounding area (Video 4, A–D). Small transport intermediates containing GPI-GFP were observed tracking into the photobleached Golgi region where they disappeared, presumably due to fusion with the Golgi complex and subsequent dispersal of fluorescence. GPI-GFP–expressing cells were labeled with STxB and CTxB and the same photobleaching protocol was followed. As shown in and , both STxB and CTxB were seen in GFP-GPI–containing structures that moved in towards the Golgi complex. We also detected additional intermediates containing STxB or CTxB but not GPI-GFP (not shown in ; see Video 4, A–D), which could represent CTxB and STxB within transferrin-containing endosomes localized near the Golgi complex. Our results indicate that the transport itinerary of GPI-GFP to the Golgi complex overlaps with that of STxB and CTxB.
GPI-GFP and a Proportion of STxB and CTxB Are Internalized via a Clathrin-independent Process
To investigate whether uptake and consequent delivery to the Golgi complex of GPI-GFP, STxB, and CTxB requires clathrin-dependent endocytic machinery, we used dominant negative mutants of epsin and eps15, proteins necessary for clathrin-mediated endocytosis (Chen et al. 1998
; Benmerah et al. 1999
). In cells transiently transfected or microinjected with a plasmid expressing epsin or eps15 mutants, transferrin uptake was reduced significantly more than uptake of CTxB and STxB ( and ; quantitation in D). This differential effect is consistent with only a proportion of the total CTxB and STxB being internalized via the same endocytic machinery as transferrin, implying that the remainder may use a clathrin-independent mechanism. Importantly, CTxB and STxB were still efficiently delivered from the PM to the Golgi complex in the absence of clathrin-mediated internalization ( C).
Figure 6 GPI-GFP, STxB, and CTxB are internalized via a clathrin-independent process. (A) Expression of a dominant negative mutant of epsin blocks transferrin-Cy3 uptake more than uptake of CTxB-Cy5. Epsin mutant was expressed by transient transfection of COS-7 (more ...)
We examined the rate of GPI-GFP cycling between the Golgi and the PM by using photobleaching techniques in cells expressing mutants of epsin. Cells were microinjected with the epsin mutant plasmid, left for 6 h to allow expression, and then the Golgi pool of GPI-GFP fluorescence was photobleached. Recovery of Golgi fluorescence in the mutant cells was no different than in noninjected cells ( E). Thus, whereas transferrin uptake requires epsin, eps15, and the clathrin-dependent endocytic machinery, delivery of GPI-GFP, STxB, and CTxB from PM to Golgi complex does not.
As a more direct test of whether GPI-GFP is taken up via clathrin-coated pits, we used immunoelectron microscopy to ascertain the distribution of GPI-GFP on the cell surface ( F). GPI-GFP was clearly excluded from coated pits and vesicles (linear density of gold particles on the PM was 4.07 ± 0.28 μ−1, in clathrin-coated pits and vesicles it was 0.07 ± 0.02 μ−1,and background [mitochondrial] signal was 0.05 ± 0.02 μ−1).
GPI-GFP, STxB, and CTxB Do Not Require Rab5 Activity for Delivery to the Golgi Complex
Early endosome function requires the GTPase rab5, and expression of the GDP-bound form of rab5 (S34N mutant) perturbs early endosomes and inhibits transferrin uptake (Stenmark et al. 1994
). In cells expressing rab5 S34N, transferrin uptake was significantly reduced, whereas delivery of CTxB and STxB (not shown) to the Golgi complex was not significantly affected ( A). Moreover, selective photobleaching of the Golgi pool of GPI-GFP in cells expressing rab5 S34N did not reveal any change in the kinetics of exchange of GPI-GFP between PM and Golgi pools ( B). Thus, the activity of rab5 is not required for delivery of raft markers from the PM to the Golgi complex.
Figure 7 GPI-GFP, CTxB, and STxB do not required rab5 activity for delivery to the Golgi complex. (A) Expression of a dominant negative mutant of rab5 (S34N) blocks intracellular accumulation of transferrin-Cy3 without preventing delivery of CTxB-Cy5 to the Golgi (more ...)
The GTP-bound form of rab5 (Q79L mutant) causes pronounced changes in the morphology of rab5-positive endosomes, without blocking transport of transferrin through these structures (Stenmark et al. 1994
). The characteristic morphology of early endosomes under these conditions allowed us to ask whether any GPI-GFP whatsoever is found in such structures. As shown in C, not even trace amounts of GPI-GFP were found in the enlarged early endosomes produced by rab5 Q79L.
Effect of 20°C Incubation on PM–Golgi Cycling
Incubation at 20°C blocks both uptake of STxB to the Golgi complex and exit from this organelle (Mallard et al. 1998
; Van Deurs et al. 1988
). We found that uptake of transferrin was not significantly perturbed at this temperature, but both CTxB ( A) and STxB (not shown, but see Mallard et al. 1998
) were only taken up into transferrin-containing endosomes, not into the Golgi complex. This led us to test whether the PM–Golgi trafficking of raft markers is differentially sensitive to 20°C treatment. When the Golgi pool of GPI-GFP was photobleached in cells held at 20°C for 40 min, recovery of this pool was effectively blocked ( B), showing that transport of GPI-GFP from the cell surface into the Golgi complex occurs very inefficiently, if at all, under these conditions. Therefore, the PM–Golgi cycling of raft markers appears to be more sensitive to 20°C treatment than the endocytic trafficking of transferrin.
Figure 8 Effect of a 20°C block and cholesterol depletion on GPI-GFP, STxB, and CTxB trafficking. (A) Incubation at 20°C blocks delivery of CTxB-Cy5 to the Golgi complex, but not uptake of transferrin-Cy3. GPI-GFP–expressing COS-7 cells (more ...)
Effect of Cholesterol Depletion on GPI-GFP, STxB, and CTxB Trafficking
Drugs that perturb cholesterol activity are known to inhibit traffic of markers for lipid rafts during both exocytosis and endocytosis, and have been used extensively as tools to differentiate between raft- and nonraft-mediated processes (Keller and Simons 1998
; Orlandi and Fishman 1998
). Filipin, which binds to cholesterol in membranes, had little or no effect on the uptake of either CTxB or STxB to the Golgi complex at a concentration of 1 μg/ml (compare C with 4 F; in all cases intracellular STxB labeling was indistinguishable from that with CTxB). At a higher concentration of filipin, 10 μg/ml, however, internalization of both CTxB and STxB was reduced and both markers were largely excluded from the Golgi complex. The CTxB and STxB that internalized under these conditions colocalized extensively with transferrin ( C). The amount of transferrin taken up into the cells appeared normal at both filipin concentrations, though treatment with 10 μg/ml filipin caused a slightly increased accumulation in peripheral as opposed to perinuclear endosomes. Thus, cholesterol sequestration by filipin inhibits uptake of STxB and CTxB into the Golgi with little or no effect on their uptake into transferrin-containing endosomes.
To test the effect of cholesterol perturbation on GPI-GFP transport, GPI-GFP–expressing cells were treated with 10 μg/ml filipin for 60 min and examined. GPI-GFP had partially redistributed from the cell surface to the Golgi complex under these conditions ( D). This redistribution most likely occurred as a result of endocytosis, as it occurred similarly in cycloheximide-treated and untreated cells, and as total GFP fluorescence in filipin-treated cells did not increase (total fluorescence was 80 ± 7%, n = 14, of starting fluorescence after 1 h in 10 μg/ml filipin). After 60 min at the lower concentration of filipin (1 μg/ml), accumulation of GPI-GFP in the Golgi complex was also apparent ( D). This contrasts with the lack of an effect on CTxB or STxB at this filipin concentration ( C), implying that exit of GPI-GFP from the Golgi complex is more sensitive to filipin treatment than uptake of STxB and CTxB (and potentially GPI-GFP) from the cell surface. These findings show that the trafficking of GPI-GFP, STxB, and CTxB is more sensitive to cholesterol depletion than the trafficking of transferrin.