25. Baxter-Burrel, A., Yang, Z., Springer, P. S. & Bailey-Serres, J. RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 296, 2026���2028 (2002). 26. Lacombe, B., Pilot, G., Gaymard, F., Sentenac, H. & Thibaud, J.-B. pH control of the plant outwardly- rectifying potassium channel SKOR. FEBS Lett. 466, 351���354 (2000). Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We thank H. Hofte, �� H. Sentenac and C. Vander Willigen for critical reading of the manuscript N. Declerk for discussions Y. Boursiac for help with cell pressure probe measurements and J. B. Thibaud and C. Plassard for assistance in micro-electrode pH measurements. This work was supported in part by the Centre National de la Recherche Scientifique (Action Thematique �� Incitative sur Programme et Equipe ���Function and regulation of plant aquaporins���). M.S. is on leave from the Laboratorio de Biomembranas, Departamento de Fisiolog����a, Facultad de Medicina, Universidad de Buenos Aires, Argentina. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to C.M. (maurel@ensam.inra.fr). .............................................................. ER���phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells Pierre Guermonprez1*, Loredana Saveanu2*, Monique Kleijmeer3, Jean Davoust4, Peter van Endert2 & Sebastian Amigorena1 1INSERM U520, Institut Curie, 26 rue d���Ulm, 75005 Paris, France 2 INSERM U580, Institut Necker, 161 rue de Sevres, ` 75015 Paris, France 3Department of Cell Biology, UMC Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands 4CNRS UMR8115, Genethon, 1 bis rue de l���Internationnale, 91002 Cedex 02, Evry, France * These authors contributed equally to this work ............................................................................................................................................................................. Induction of cytotoxic T-cell immunity requires the phagocytosis of pathogens, virus-infected or dead tumour cells by dendritic cells1. Peptides derived from phagocytosed antigens are then presented to CD81 T lymphocytes on major histocompatibility complex (MHC) class I molecules, a process called ���cross-pres- entation���2,3. After phagocytosis, antigens are exported into the cytosol and degraded by the proteasome4���6. The resulting pep- tides are thought to be translocated into the lumen of the endoplasmic reticulum (ER) by specific transporters associated with antigen presentation (TAP), and loaded onto MHC class I molecules by a complex ���loading machinery��� (which includes tapasin, calreticulin and Erp57)7. Here we show that soon after or during formation, phagosomes fuse with the ER. After antigen export to the cytosol and degradation by the proteasome, pep- tides are translocated by TAP into the lumen of the same phagosomes, before loading on phagosomal MHC class I mol- ecules. Therefore, cross-presentation in dendritic cells occurs in a specialized, self-sufficient, ER���phagosome mix compartment. The reasons why phagocytosis itself favours cross-presentation in dendritic cells and macrophages are unclear. To gain further insight into this process, we purified phagosomes from immature dendritic cells. After purification, the enrichment of various phagosomal markers was analysed by western blotting. Early phagocytic mar- kers, such as transferring receptor and phosphoinositide-3-kinase (Pi3Kp85), were detected early after engulfment and rapidly decreased thereafter. Markers of the late endocytic pathway, such as rab7 (Fig. 1a) and Lamp2 (Fig. 1b, left panels) increased over time, indicating phagosome maturation towards phagolysosomes. In macrophages, phagosomes were recently shown to fuse with the ER during or soon after engulfment8. Similarly, we found that several ER resident proteins such as sec61/sec62 and calnexin were detected immediately after phagosome formation in dendritic cells (Fig. 1a and Fig. 1b, right panels). Recruitment of ER residents into phagosomes, however, was restricted to a subset of the ER proteins detected by polyclonal antibodies raised against whole ER mem- branes (Supplementary Information SD1). The presence of these ER markers in phagosomes decreased as phagosomes matured into phagolysosomes. To quantify the proportion of phagosomes bearing ER markers, we used flow cytometric (FACS) analysis of phagosomes9. After the pulse with latex beads, only 10% of the phagosomes expressed Lamp2, and the proportion of Lamp2-bearing phagosomes increased over time (Fig. 1b, left panel), to reach over 50% after 240 min chase. The distribution of calnexin, in contrast, was homogeneously high in early phagosomes (right panels in Fig. 1b), suggesting that most of the phagosomes had already fused with ER membranes. Consistent with our biochemical analysis and with previous results in macrophages8, calnexin labelling of phagosomes decreased over time. ER recruitment to phagosomes was also shown in whole cells, using confocal-laser scanning-microscopy (CLSM). In immature dendritic cells pulsed with fluorescent beads, staining of the phago- somal membrane was observed using both total ER antibodies (not shown) and calnexin antibodies (Fig. 1e), in a wide proportion of early phagosomes. Electronic microscopy analysis of ER distri- bution by calreticulin staining demonstrates the juxtaposition of ER membranes to the forming phagosomes (phagocytic cups were stabilized by PI3K inhibitors8), and strongly suggests direct fusion between the two organelles (Fig. 1c). Direct evidence for ER���phagosome fusion was obtained using a cytochemical EM technique, revealing the activity of the ER enzyme glucose-6- phosphatase (G-6-Pase) on Epon sections10. G-6-Pase activity appears as dense precipitates in numerous ER structures through- out the cells, including the nuclear envelope (Fig. 1d). Strikingly, the majority of phagosomes displayed strong G-6-Pase labelling some- times over the whole of the limiting membrane (Fig. 1d, right panel), other times on part of it (Fig. 1d, left panel). We concluded that in dendritic cells, like in macrophages8, phagosomes fuse with the ER, soon after or during particle engulfment. ER proteins then progressively disappear as phagosomes mature into phagolysosomes. Fusion with the ER and the recruitment of ER proteins to phagosomes led us to reconsider the conventional view of cross- presentation. We hypothesized that after antigen export to the cytosol and degradation by the proteasome, peptides could be translocated back into the lumen of the same phagosome by TAP, and loaded onto MHC class I molecules in the phagosomal lumen. To test this model, we first examined whether ER fusion with phagosomes actually results in recruitment of TAP and of the MHC class I loading machinery. Abundant TAP was detected in early phagosomes and was reduced over time (Fig. 2a, right panel). Flow cytometric analysis of phagosome populations9 of various ages revealed homogeneous staining of early phagosomes, which also decreased over time (Fig. 2a, left panel). EM analysis of purified phagosomes showed that TAP2 is inserted into the phagosomal membrane surrounding the latex bead (Fig. 2b). The recruitment of TAP to phagosomes was also seen in intact dendritic cells using CLSM (Fig. 2c). In spite of high staining of abundant ER mem- branes throughout the cells, enrichment in membranes surround- ing the beads was evident in most phagosomes. Together with TAP, tapasin, calreticulin, ERp57 and the heavy chain of MHC class I were all detected in purified early phagosomes by western blotting (Fig. 2d). A clear decrease over time was observed for tapasin and ERp57, whereas MHC class I heavy chain and calreticulin remained stable. Therefore, in the first few letters to nature NATURE | VOL 425 | 25 SEPTEMBER 2003 | www.nature.com/nature 397 �� 2003 Nature Publishing Group

hours after engulfment, phagosomes recruit the MHC class I peptide translocation and loading machinery. We next examined whether cross-presentation of phagocytosed antigens occurs in the same time window as ER recruitment to phagosomes that is, in the first 2���3 h after phagocytosis. The egress of antigens from phagosomal lumen to the cytosol is detected after 1.5 h (Supplementary Information SD2). Consistently, cross- presentation of the OVA257���264 peptide (SIINFEKL) by the H-2Kb class I MHC molecule reached a plateau within 3 h of chase (Fig. 3a). At these early time points, cross-presentation of OVA-coated Figure 1 Rapid recruitment of ER at the early stages of phagosome biogenesis in immature dendritic cells. a, Western blot analysis of phagosomal maturation. tcl, total cell lysate. b, FACS and western blot (WB) analysis of calnexin and Lamp2 recruitment on phagosomes. c, Cryoelectron microscopy analysis of ER���phagosome fusion. Calreticulin- positive (gold beads) ER membranes are observed in direct apposition to the latex beads (LB) phagosomes, suggesting fusion of the two compartments (arrows). d, EM G-6-Pase staining of ER and phagosomal membranes. Phagosomal membranes containing (black arrows) or not (white arrows) G-6-Pase precipitates. e, CLSM analysis of ER recruitment at early stages of phagosome biogenesis. Asterisks indicate beads, arrows indicate the accumulation of calnexin staining around beads. Ctl, control. letters to nature NATURE | VOL 425 | 25 SEPTEMBER 2003 | www.nature.com/nature 398 �� 2003 Nature Publishing Group