In order to isolate early endosomes originating from CIE we considered ways to affinity purify this endosomal compartment. In another study ongoing in the laboratory, we had identified PM SNARE proteins that travel along the CIE pathway. Expression of an amino terminally GFP-tagged syntaxin 3 localized to the PM and tubular endosomal membranes () that also contained internalized MHCI (not shown). GFP-syntaxin 3 also localized to the vacuolar membranes containing CIE cargo proteins that form in cells expressing Arf6Q67L () (5
). Endogenous syntaxin 3, detected with a specific antibody, also localized to these endosomal membranes (not shown). Since the GFP tag was on the amino terminus of Syntaxin 3 and hence was exposed on the outer surface of the vacuoles, we decided to affinity purify these vesicles with antibodies to GFP.
Figure 1 Immunoisolation of Arf6Q67L-induced vacuoles and cargo protein identification. A. Localization of GFP-syntaxin 3 (GFP-syn3) in Hela cells alone (left) and in cells co-expressing Arf6Q67L (right). Bar, 10 μm. B. Coomassie blue staining of cell (more ...)
Whole cell lysates from HeLa cells that had been transfected with Arf6Q67L and GFP-Syntaxin 3 were incubated with anti-GFP-coupled magnetic beads to pull-out GFP-containing membranes. Although the GFP-Syntaxin 3 in cells expressing Arf6Q67L was present both at the PM and on the vacuoles, experiments with disrupted cells that had been surface biotinylated indicated that the PM fragments would reseal with the extracellular surface out and so the GFP tag would not be available for immunoisolation. Material bound to the beads was resolved by electrophoresis and the proteins stained using a sensitive Coomassie staining technique (27
). It was clear that there were many more proteins associated with the Anti-GFP beads than the IgG control beads (). Slices of gels were made and digested with trypsin as described in Materials and Methods. The subsequent peptide fragments were subjected to LC-MS/MS for identification.
Among the proteins identified through this proteomic screen were 6 new membrane proteins that might represent new cargo molecules that travel along this CIE pathway. They are : CD44, CD55, CD98, CD147, the non-insulin stimulated glucose transporter 1 (Glut1), and the intercellular adhesion molecule 1 (ICAM1) (). For three of these (CD98, CD147 and Glut1) we obtained antibodies able to recognize the endogenous protein in immunoblots. When the isolated membrane fractions were probed with these antibodies, bands representing CD98, CD147 and Glut1 were apparent in the fractions from the Anti-GFP beads and also in the whole cell lysate (). MHCI, a protein known to follow this CIE pathway (5
), also was identified in the proteomic analysis and we detected MHCI protein in the material bound to the anti-GFP-beads ().
Next, we validated that these 6 membrane proteins were associated with the Arf6Q67L-generated vacuoles in cells by immunofluorescence. Hela cells were transfected with Arf6Q67L and a GFP-tagged membrane marker (Mem-GFP), composed of GFP with the carboxyl tail segment from H-Ras, which we have previously showed associates with membranes of the CIE pathway in HeLa cells (28
). After transfection, the cells were fixed and then localization of the endogenous proteins was determined. With antibodies directed to each of the newly identified proteins, we observed that CD44, CD55, CD98, CD147, Glut1 and ICAM1 all colocalized with Mem-GFP on discrete vacuoles in the periphery (, insets) and on clustered vacuoles in the juxtanuclear region, typical of cells expressing Arf6Q67L (5
Figure 2 New cargo proteins localize to Arf6Q67L vacuoles. HeLa cells co-expressing Arf6Q67L and Mem-GFP were fixed and then distribution of the cargo proteins was revealed with antibodies directed against CD44, CD55, CD98, CD147, Glut1, and ICAM1, followed by (more ...)
An interesting aspect of these membrane proteins is that many of them either are nutrient transporters or are involved in regulating cellular interactions with the matrix, an activity that has been associated with Arf6-regulation of CIE pathway trafficking (18
). CD44 is a cell adhesion molecule and receptor for hyaluronan, an extracellular glycosaminoglycan. It is believed to direct matrix metalloproteinases to the leading edge of cells (29
). CD55, also known as decay acceleration factor (DAF) is a GPI-anchored protein as is CD59, which we previously identified as being associated with these endosomes. Both of these GPI-anchored proteins protect cells from complement-mediated lysis (30
CD98 is a multifunctional protein that is responsible for nutrient uptake and interacts with proteins involved in cell-matrix interactions (31
). It is the heavy chain of some amino acid transporters, in particular with Lat1, responsible for neutral amino acid transport. Although we also identified Lat1 in the proteomic analysis, we have not found an antibody suitable for immunolocalization. CD98 may also associate with Glut1, the ubiquitous glucose transporter, which we identified in the screen. CD98 also associates with β1-integrin, a previously identified cargo (8
), and with CD147, which is a new cargo protein identified here. CD147, also known as Emmprin for extracellular matrix metalloproteinase inducer, associates with the monocarboxylate transporter proteins that transport lactate and pyruvate across the PM (32
Having validated that these endogenous membrane proteins were in fact associated with the isolated vacuoles that were analyzed by mass spectroscopy, we next examined whether these proteins were present on CIE endosomes in untransfected cells. First, we observed that the total, steady state distribution of these cargo proteins colocalized with MHCI in HeLa cells (data not shown). Next, we used antibody internalization to assess whether these proteins followed a membrane trafficking pathway similar to that of MHCI and distinct from that of transferrin. We and others have previously shown that using monoclonal antibodies to follow the internalization of PM proteins does not alter their trafficking (5
). The antibodies do not cause cross-linking of the proteins; in fact, for MHCI, cross-linking of the primary antibodies using secondary antibodies inhibits endocytosis (unpublished observations). After 60 min internalization, CD55 localization was almost identical to that of MHCI on punctate structures in the peripheral and juxtanuclear region some of which colocalized with transferrin (). Additionally, CD55 and MHCI were both associated with the tubular, recycling endosomes that emanated from the juxtanuclear region that were devoid of transferrin (, inset) and are characteristic for the CIE pathway in HeLa cells (21
). Since we did not have antibodies that recognized the extracellular portion of Glut1, to follow Glut1 endocytosis, we transfected HeLa cells with a Glut1 construct with an extracellular HA tag (HA-Glut1) so we could examine internalization with anti-HA antibodies. After 60 min internalization, HA-Glut1 colocalized with the internalized MHCI on some perinuclear punctate structures that colocalized with transferrin and on tubular endosomes devoid of transferrin ( inset) as we observed with CD55. The distribution of HA-Glut1 was similar to that of endogenous Glut1, which colocalized with endocytosed MHCI on juxtanuclear endosomes and tubular recycling endosomes (Supp. Fig. 1
). Although we could assess the steady state distribution of ICAM1 in cells by immunofluorescence (), we were unable to follow endocytosis of ICAM1 using the antibodies available.
Figure 3 New cargo proteins differ in their intracellular trafficking. Cells were incubated with monoclonal antibodies directed towards CD55, HA-Glut1, CD44, CD98 and CD147 in the presence of antibodies to MHCI and labeled transferrin for 60 min. For A, unlabelled (more ...)
In contrast to what we observed with CD55 and Glut1, CD44, CD98 and CD147 showed a different pattern of internalization. CD44 colocalized with MHCI on tubular endosomes but was not associated with MHCI in the juxtanuclear region nor did it colocalize with transferrin (). CD98 and CD147 distributions were especially striking as they were prominently associated with tubular endosomal membranes and absent in the juxtanuclear region where MHCI colocalized with transferrin ( inset). This different distribution after 60 min of internalization suggested some differences in itinerary for these new cargo proteins. To examine this more closely we followed the internalization of CD98 and MHCI over shorter periods of time. At 5 min, CD98 and MHCI were observed in the same punctate structures and then at 10 min CD98 was observed in tubular endosomes but MHCI did not appreciably reach these structures until 30 min ().
Figure 4 CD98 and MHCI colocalize at short times of internalization. HeLa cells were incubated with directly conjugated monoclonal antibodies to CD98 and MHCI for 5, 10 and 30 min and then surface antibody was removed by acid wash prior to fixation. Bar, 10μm. (more ...)
In addition to CD98, we noticed that CD44 and CD147 also appeared to gain access to the tubular endosome more rapidly than did MHCI, possibly by evading fusion with the common “classical” early endosome that contained transferrin, Rab5 and the early endosomal antigen 1 (EEA1). To test this possibility, we examined whether internalized cargo proteins reached endosomes that labeled with EEA1. After 30 min internalization, CD55 and HA-Glut1 were observed in puncta in the juxtanuclear region that colocalized with EEA1, similar to that of MHCI (), as we have reported previously (5
). In contrast, internalized CD44, CD98 and CD147 were predominantly associated with endosomal tubules and vesicles that were devoid of EEA1 (). These findings indicate that CD44, CD98 and CD147 largely avoid whereas CD55, Glut1 and MHCI readily reach EEA1- and transferrin-containing early endosomes. Thus, although all cargo proteins entered by CIE and became trapped in Arf6Q67L-associated endosomes, CD44, CD98 and CD147 appeared to by-pass EEA1 and transferrin-containing endosomes on their way to the tubular recycling endosome.
Figure 5 CD55, Glut1 and MHCI reach, whereas CD98, CD147 and CD44 do not reach, EEA1-positive compartments. Unlabeled antibodies to CD55, HA, MHCI, CD98, and CD147 were incubated with cells for 30 min to allow internalization. After removal of free antibody, cells (more ...)
Since the itinerary of some of these new cargo proteins differed from that of MHCI, we followed the itinerary of CD98 in more detail to confirm that this new type of cargo protein was still utilizing the same CIE pathway that we had characterized for MHCI and CD59 (5
). In particular, we examined whether internalization of CD98 was independent of dynamin and dependent on PM cholesterol and whether the trafficking of CD98 was altered upon expression of mutants of Rab22. Expression of the K44A mutant of dynamin2 did not affect internalization of CD98 or MHCI whereas transferrin internalization was inhibited (Supp. Fig. 2A
). Additionally, treatment of cells with filipin to bind cell surface cholesterol (9
) blocked internalization of CD98 (Supp. Fig. 2B
) and MHCI as previously described (9
). We previously showed that Rab22 is associated with the tubular recycling endosome associated with this CIE pathway in HeLa cells and that expression of the active mutant of Rab22 (Q64L) enhanced the tubular phenotype and also resulted in the accumulation of vesicles at the distal ends of the tubules (21
). Furthermore, these tubules were absent in cells expressing the S19N dominant negative form of Rab22. We found that internalized CD98 and MHCI both colocalized with GFP-Rab22 and that in cells expressing Rab22Q64L there was an increase in number of cells exhibiting tubules and peripheral vesicles (Supp. Fig. 3
) as we had observed previously for MHCI (21
). Additionally in cells expressing Rab22S19N there was a marked decrease in number of cells exhibiting tubules and the internalized CD98 and MHCI colocalized in a perinuclear region of the cell (Supp. Fig. 3
), which also contains transferrin receptor (21
). Although our previous studies showed that expression of Rab22S19N inhibits recycling of MHCI but not that of transferrin (21
), we were not able to quantitatively measure recycling of CD98 with the reagents at hand. We suspect, however that once in the tubular endosome, the recycling of the new cargo proteins is dependent upon the same components (Rab22, Rab11 and Arf6) that regulate recycling of MHCI (21
Taken together, these PM proteins appear to be new CIE cargo that enter cells independently of dynamin, but dependent upon PM cholesterol and accumulate in Arf6Q67L vacuoles. Once inside the cell, however, the new cargo proteins revealed differences in their itineraries (). CD55 and Glut1 followed the same pathway as that of internalized MHCI ( Red Bars) and could be observed in endosomes that contain EEA1 and transferrin and then later in the tubular recycling endosomes. This also appears to be the route traveled by CD59 (9
), integrins (8
) and syndecan (7
). In contrast, CD44, CD98 and CD147 ( Green Bars) did not appear to enter the EEA1-positive compartment but appeared to join recycling tubular endosomes by way of the juxtanuclear endocytic recycling compartment (ERC). Previously we had observed evidence for this more direct recycling route taken by CD44, CD98 and CD147 when we were examining the distribution of Arf6, H-Ras and certain PM SNARE proteins. In HeLa cells these proteins are present on Arf6Q67L vacuoles and on the recycling tubules but never on enlarged, Rab5Q79L endosomes (5
). We now have identified the cargo proteins that follow this direct pathway (CD44, CD98, CD147) in contrast to those cargo proteins that also merge with the EEA1 and Rab5 endosome (Glut1, CD55, CD59 and MHCI). We are in the process of identifying which features in cargo proteins might route them towards EEA1 compartments. Despite the different routes to the tubular endosome, both types of cargo are nonetheless trapped in the same structures, presumably the ERC, in cells expressing Rab22S19N.
Figure 6 Model showing possible sorting within the CIE pathway in HeLa cells. Trafficking of CIE (red and green bars) and CDE (blue bars) cargo proteins. Some CIE cargo (red bars) traffics along with MHCI to EEA1 early endosomes en route to late endosomes (LE) (more ...)
The new CIE cargo proteins identified here join those already known and together they begin to reveal the physiological functions of this endocytic pathway. A number of these proteins (CD44, CD98, CD147, integrins and syndecan) are involved in cell-matrix interactions and are implicated in cell migration and metastasis. Another set of proteins are nutrient and ion transporters (CD98, CD147, Lat1, Glut1, potassium channels, mucolipin-2) or proteins that present themselves to the immune system (MHCI, Cd1a and MHCII). Additionally, we recently showed that the β2 adrenergic and M3 muscarinic receptors constitutively traffic along this CIE pathway and then upon ligand activation switch to internalization by clathrin pathways (16
). The trafficking of these plasma membrane proteins and in particular recycling of these proteins to particular regions of the cell could have important consequences during development and for cell homeostasis. These proteins all lack known adaptor protein binding motifs but may contain other sequences in their trans-membrane or cytoplasmic regions that could interact with alternative coats or machinery used during CIE. With this new set of tools, we can examine CIE pathways and follow the trafficking of endogenous cargo proteins in different cell types to see how they are organized. These studies will likely reveal the range of CIE pathways in different cell types and provide insight into the cellular functions of such pathways.