On the other hand, in cells expressing hRab22aQ64L, Tfn localized in large hRab22a-containing endosomes in the periphery of the cell (A to C). in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized Shikimic acid (Shikimate) the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes. Most macromolecules internalized by clathrin-dependent and -independent endocytosis are delivered to sorting endosomes, a dynamic tubovesicular compartment with a central role in the targeting of material to different intracellular destinations (10). Within this compartment, a first segregation of membrane-bound from soluble molecules occurs by geometrical means. The narrow tubules pinching off from sorting endosomes are enriched in membrane-associated molecules because of their large area-to-volume ratio. In contrast, soluble molecules (e.g., ligands dissociated from their receptors at the low pH of endosomes) accumulate in the vesicular portion of sorting endosomes. Most of the material associated with tubules is either transported back to the plasma membrane or transferred to the recycling compartment. In contrast, most of the soluble material is delivered to late endosomes. Membrane invaginations in the sorting endosomes form intraluminal vesicles that are also targeted to late endosomes, providing an efficient route to digest transmembrane proteins (e.g., receptors that need to be down-regulated) (7, 25). Sorting, late, and recycling endosomes also exchange macromolecules with the exocytic pathway, mostly with the for 15 min at 4C. Protein concentration in lysates was determined using the bicinchoninic acid protein assay (Pierce Biotechnology Inc., Rockford, IL). The supernatants containing the cytosolic fraction of proteins were Shikimic acid (Shikimate) separated on 12% sodium dodecyl sulfate-polyacrylamide gels (25 g protein per lane) and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Nonspecific binding was blocked by incubation of the membranes with 5% nonfat milk, 0.5% Tween 20, and PBS for 1 h at 37C. The primary and secondary antibodies were diluted in blocking buffer and incubated overnight at 4C and 1 h at room temperature. After each incubation step, the blots were washed five times for 7 min with 0.5% Tween 20 and PBS. Bound primary antibodies were visualized using peroxidase-conjugated secondary antibodies and an ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Modeling of Tfn transport. Differential equations for Tfn transport in a two-endosomal compartment model have been described previously (see reference 21 and the supplemental material therein). Data were fitted to the model by minimization of [(predicted values ? observed values)2]. Rate Shikimic acid (Shikimate) constants were iteratively adjusted during the minimization process. These constants were defined as described previously (21). Statistical comparison of fits was performed by Fisher’s F test as described in reference 14. RESULTS In CHO cells, the TfnR is trapped in Rab22a-containing vesicles. Three major routes are followed by proteins and lipids exiting sorting endosomes. Some macromolecules are directed to lysosomes, others are directed to the TGN, and another set is recycled back to the cell surface. We have observed that expression of GFP-cRab22a in CHO cells causes a striking enlargement of the sorting endosomes (13). In these cells, transport to lysosomes is not disrupted. In contrast, a remarkable delay in transport from endosomes to the TGN was observed (12). We wondered whether cRab22a Rabbit Polyclonal to OR5I1 expression affects the recycling of macromolecules to the cell surface, the other major route. Specifically, we were interested in Tfn recycling that has been well characterized in TRVb-1 cells, a CHO cell line stably expressing the human TfnR. According to published results, in TRVb-1 cells, TfnRs localize predominantly in the recycling center, and a minor percentage is present in early endosomes scattered.