The identity of organelles is

The identity of organelles is defined by a particular set of molecules present on their surface. Most organelles in the endomembrane system (i.e., endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes) carry Rab GTPases (hereafter referred to as Rabs), which serve as binding platforms for the unique localization of protein machinery to a particular organelle. This machinery functions, for instance, in the transport of vesicles, such as Rab-bound motor proteins 3, 4, 5, 6, 7, or in the recognition of a vesicle at the acceptor organelle, such as Rab-interacting tethering molecules 8, 9, 10, 11. In addition, signaling lipids, such as defined phosphoinositides, provide a second layer of identity and recruit organelle-specific proteins. The endocytic pathway not only allows cells to take up material from the outside, but also functions as a major pathway of membrane protein recycling, organelle quality control, and signaling 12, 13. It connects the plasma membrane with downstream organelles, the endosome and lysosome (Figure 1). Endocytosis starts with the collection of cargo proteins at the plasma membrane, the formation of an endocytic vesicle, and its fission (see Glossary). These vesicles fuse with the early endosome, a major sorting station for membrane proteins and probably lipids, given that they are major constituents of the vesicle (Box 1). Once plasma membrane receptors, such as the LDL receptor, have released their cargo into the endosomal lumen, they are collected in tubular carriers and transported back to the plasma membrane, making them available for another round of endocytosis [14]. Their cargo remains in the lumen of the organelle. Membrane proteins, such as ion or amino Physostigmine hemisulfate transporters, are marked with ubiquitin by specific ubiquitin ligases at the plasma membrane, endocytosed, and sorted at the endosome into intraluminal vesicles. These intraluminal vesicles accumulate in the lumen of endosomes. Via these processes, endosomes consequently change their composition and morphology, and convert from a tubular organelle into a round multivesicular body, also termed the late endosome (Box 2). This late endosome eventually fuses with the lysosome, thereby releasing the intraluminal vesicles into the lumen of the lysosome [13]. This results in the hydrolysis of all delivered macromolecules into amino acids, lipids, and carbohydrates for further use by the cell (Box 1 and Figure 1). Several additional pathways feed into this endolysosomal pathway. For example, autophagosomes or Golgi-derived, AP-3-coated vesicles also fuse with endosomes or lysosomes [15]. Organelles need to maintain their lipid and protein composition for the sake of their identity and functionality, since multiple events of unidirectional vesicle-mediated transport would lead to a mixing of organellar content and lipid composition. The balance is achieved in part by retrograde transport pathways between organelles, which compensate for the membrane flux and return receptor proteins to their starting position 16, 17. Rabs are considered to be markers of membrane identity and function as central organizers of vesicle-mediated transport between organelles (Figure 2A) [18]. Rabs, a subfamily of the Ras superfamily of small GTPases, are highly conserved in eukaryotic cells. Human cells express approximately 70 different Rabs, whereas yeast expresses 11 members of this family. Rabs can switch between an active GTP-bound state (Rab-GTP) and an inactive GDP-bound form (Rab-GDP). This switch requires two regulatory proteins, guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). All Rabs are prenylated at their C termini and maintained in soluble form in the cytosol by binding to the GDP dissociation inhibitor (GDI). To function on membranes, they require a GEF as a Rab-specific activator, which triggers GDP release and loading of the more abundant GTP [1]. Hence, GEF-mediated nucleotide exchange stabilizes the Rab on membranes. GEFs bind to organelle-specific cues, such as phosphoinositides, cargo proteins, or other Rabs, and, thus, trigger the local recruitment and activation of their cognate Rab [19]. Even though additional factors have been implied in Rab recruitment to membranes, GEF localization appears to lie at the heart of correct Rab localization 1, 8, 10. GTP binding stabilizes two loops in the Rab GTPase domain and allows the Rab to recruit specific effector proteins to the membrane [20]. Rabs are incomplete enzymes, and depend on a specific GAP to trigger GTP hydrolysis. GDI is unable to bind to GTP-bound Rabs, yet has binding sites for both the Rab-GDP and its lipid anchor, and, thus, can extract the Rab-GDP from membranes and make it available for another round of membrane delivery and activation [21]. In this review, we focus on the endocytic pathway to provide insights into how organelle identity is established and maintained, and how identity markers are used to organize specific tethering and fusion events. While not focusing on these, such an understanding is critical for any intervention during infection processes that abuse the endocytic system or drug delivery or enzyme replacement via the endocytic pathway.