Endocytosis is the process by which membrane proteins are removed from the cell surface by internalization (Jovic et al., 2010
; Maxfield and McGraw, 2004b; Traub, 2009
). It is crucial for rapid and localized regulation of levels of proteins at the plasma membrane. After being endocytosed, transmembrane proteins can be either degraded or recycled back to the cell surface. Furthermore, the endosomal system is also important for intracellular signaling since internalized receptors continue to transmit signals from endosomes that can be distinct from those that immediately arise at the plasma membrane (Maxfield and McGraw, 2004a
; Murphy et al., 2009
). The endosomal system is most thoroughly studied in non-polarized cells. “Classical” endosomal system contains three major types of compartments: early endosomes (EE), recycling endosomes (RE) [also frequently referred to as ERC = endosomal recycling compartment], and late endosomes (LE)/lysosomes (Lys). Membrane proteins are first endocytosed into EEs. From there, they can either follow the degradative pathway to LE/Lys or recycle back to the plasma membrane either directly from EE or through the recycling endosome RE (). Owing to the advancement in imaging techniques, especially high-resolution live cell imaging, it is now clear that the endosomal system is very dynamic and more complex than previously anticipated. In contrast to the long-held view that EEs are the initial cargo sorting station, live cell imaging demonstrated that various membrane receptors can be sorted prior to entering EEs. Cargo sorting starts already at the plasma membrane, at the level of clathrin-coated pit (CCP) formation, such that different cargo is accumulated in different CCPs. Endocytosed G protein-coupled receptors (GPCRs) accumulate in CCPs with prolonged surface residence time in comparison to transferrin receptors (TfR) which are internalized by short-lived CCPs (Puthenveedu and von Zastrow, 2006
). As a consequence, distinct cargos are sorted into different types of early endosomes. Several cargos en route to degradation are preferentially targeted to dynamic EEs, whereas the recycling cargo transferrin is enriched in a larger, static population of EEs (Lakadamyali et al., 2006
). The exact mechanisms and machinery responsible for these sorting events is not well understood.
Model of neuronal endomembrane system
Another layer of complexity was recently observed in sorting within EEs. It was previously shown that recycling cargo sorts to the tubular subdomain of early endosome and later to tubular REs, whereas degradative cargo remains in the vacuolar portion of EEs (Mayor et al., 1993
). It was also shown that within REs different cargos are transported in distinct carriers with different transport kinetics (Lampson et al., 2001
). Now, live cell imaging demonstrated that tubular microdomains on endosomes are heterogeneous, with dynamic tubules sorting constitutively recycling transferrin and more stable tubules sorting sequence-dependent recycling β2 adrenergic receptor (Puthenveedu et al., 2010
). Generally, there are more flavors to each type of endosomal compartment than we anticipated. Furthermore, certain cargos also traffic retrogradely from REs to the TGN (Ghosh et al., 1998
; Mallard et al., 1998
), and REs are on the anterograde, biosynthetic route from the TGN to the plasma membrane of some cargos in epithelial cells (Ang et al., 2004
; Folsch et al., 2009
). There is clearly a close and dynamic relationship between REs and the TGN (Schmidt and Haucke, 2007
), making the distinction between secretory and endosomal pathways more difficult. This complexity is presumably crucial for precise sorting of various receptors depending on their destinations.
Along with the segregation of cargos to different endosomal compartments, there is also segregation of endosomal regulators (Miaczynska and Zerial, 2002
; Stenmark, 2009
), which are crucial for cargo trafficking and the maintenance of specific endosomal compartments. These regulators belong to multiple different families of proteins such as: 1) small GFP-ases (rabs) and their interactors such as EEA1 and APPL, 2) adaptor proteins (AP-1, -2, -3, -4), that recognize cargo sorting motifs and recruit coat proteins important for vesicle formation 3) SNARE proteins involved in vesicle fusion, 4) exocyst complex involved in exocytosis, 5) EHD proteins proposed to be involved in tubulation and fission processes, 6) BAR domain-containing proteins which cause membrane bending, 7) sorting nexins containing a PX domain for membrane binding, among others. Some of these regulators are transmembrane proteins (such as most of the SNAREs), but others like rabs, EHDs, EEA1 and APPL, can be recruited in a dynamic and regulated fashion to the endosomal membrane from the cytosol. EEs are defined by the presence of a subset of endosomal regulators, in particular EEA1 and rab5. REs associate with rab11, and LEs with rab7.
How endosomal identity is maintained in the face of continuous membrane and cargo flux, and how transport between specific endosomal populations is regulated is under intense investigation, particularly in non-neuronal cells. It is now well-established that individual endosomes frequently consist of heterogeneous subdomains which segregate laterally within a single endosome. For instance, EEs have distinct rab5- and rab4- subdomains, REs have rab4- and rab11-subdomains (Miaczynska and Zerial, 2002
; Sonnichsen et al., 2000
), and LEs have rab7- and rab9-subdomains (Barbero et al., 2002
). These mosaic subdomains might correspond to regions of cargo fusion into an endosome, subsequent cargo sorting into distinct domains, and ultimate budding of new endosomal transport carriers containing a subset of the total membrane cargos contained in the endosome. In addition, there is striking evidence that early compartments can mature into later compartments by shedding early regulators and gaining later regulators. This has been beautifully demonstrated by a recent paper from the De Camilli laboratory. Live cell imaging of endosomal compartments labeled with different endosomal regulators showed that pre-early endosomal compartments (APPL-positive) convert to early endosomes (EEA1-positive) (Zoncu et al., 2009
), by shedding APPL and recruiting EEA1 to the same pre-existing endosome. Subsequently, early endosomes (rab5-positive) can mature into late endosomes (rab7-positive) (Rink et al., 2005
) by shedding rab5 and recruiting rab7. Little is known about how the conversion of one compartment to another (and its concomitant switch in associated regulators) is achieved. For the switch from the APPL-positive preEE to the EEA1-positive EE, rab5 activity and accumulation of a different phosphoinositide species, PI-3P, are required (Zoncu et al., 2009
). How rab5 converts to a rab7-positive LE is not known, but rab activity and phosphoinositides likely play a role here as well.