Rab and Arf G proteins in endosomal trafficking

Abstract

Endocytosis is a fundamental process that cells use to remove receptors, extracellular material, plasma membrane proteins and lipids from the cell surface. After entry into cells, the cargo proteins are subsequently trafficked to late endosomes and lysosomes for degradation, to the Golgi complex, or to recycling endosomes for return to the plasma membrane. Small G proteins in the Rab and Arf family are present on endosomes and coordinate the trafficking of cargo proteins. Here we describe some basic experimental approaches to begin to study the endosomal trafficking of a given cell surface protein.

Introduction

Endocytosis is an important phenomenon where cells internalize different extracellular molecules, ligands, membrane proteins, and lipids. Endocytosis occurs by different mechanisms that may be dependent on or independent of the clathrin coat (Conner & Schmid, 2003). Clathrin-independent modes of entry include macropinocytosis, phagocytosis, caveolar, and a variety of other possibly distinct forms (Mayor, Parton, & Donaldson, 2014). Whatever the entry mechanism is, the cargo molecules are captured in the endosomes that are pinched off from the plasma membrane and then typically these endosomes fuse with early or sorting endosomes to begin their intracellular journey to the final destination.

Collectively, “endosomes” consist of different heterogeneous compartments that can be designated as incoming endosome, early endosome, late endosome, or recycling endosome depending on the kinetics with which they are loaded with endosomal cargo (Maxfield & McGraw, 2004). Within this endosomal system, the incoming cargo are sorted for transport to the trans Golgi network (TGN), degradation in lysosomes, or recycling back to the plasma membrane depending on cytoplasmic sorting sequences on the cargo molecules and the machinery assembled around it (Figure 1(A)). This sorting of membrane and cargo is a crucial step in the proper functioning of endosomal systems in the cell. In the degradative pathway, membrane proteins are often recognized by ubiquitination of residues in their cytoplasmic domains that facilitates sorting into multivesicular bodies and then to the lysosomes where they are degraded (Piper & Katzmann, 2007). Recycling pathways are numerous, may vary with the type of cargo and become quite complex in polarized cells. The transferrin receptor (TfR) is a prototypical cargo protein that enters through clathrin-mediated endocytosis (CME) to deliver, via its bound-transferrin, iron to the cytoplasm and is then recycled back to the cell surface via a rapid recycling pathway or a slower pathway that emanates from the juxtanuclear endocytic recycling compartment. The recycling route can also be different for cargo proteins that enter the cells by clathrin-independent endocytosis (CIE); in many cells, CIE cargo proteins recycle back to the plasma membrane via specialized recycling tubules (Grant & Donaldson, 2009). Endosomal compartments are defined by their protein and lipid composition. This includes differences in phosphoinositides (PIPs) and association with Ras family GTP-binding proteins (G proteins) (Di Paolo & De Camilli, 2006).

In eukaryotic cells, the formation of transport vesicles and their specific delivery to target membranes are often regulated by different small G proteins mainly of the Rab and Arf families. There are over 70 mammalian Rab proteins and more than half of them are involved in regulating endosomal membrane traffic (Wandinger-Ness & Zerial, 2014). Rabs define compartment identity and are involved in vesicle formation, transport, and fusion to the target compartment. These varied roles are accomplished through the many Rab effector proteins. Rab5 is a central endosomal Rab that defines the initial sorting compartment or early endosome. Rab5, through its effectors, regulates endosomal fusion, signaling, sorting, and transport along cytoskeletal elements (Zeigerer et al., 2012). Rabs 4, 7, 9, 11, 22, and 35 have all been known to play important roles in the endosomal systems. Rab4, Rab35, Rab11, and Rab22 are known to play a role in the recycling of cargo back to the plasma membrane through recycling endosomes (Kouranti et al., 2006, van der Sluijs et al., 1992, Weigert et al., 2004). Rab7 has been shown to be important for endosomal fusion and maintenance of lysosomal structures whereas Rab9 regulates cargo transport from the late endosomes to the TGN (Wandinger-Ness & Zerial, 2014). Rab G proteins act as molecular switches in regulating different cellular events by their activation and inactivation.

The Arf family of G proteins also control membrane traffic through a regulated cycle of GTP-binding to activate and GTP-hydrolysis to inactivate the Arf protein. Arfs localize to membranes throughout the cell, including the plasma membrane and the membranes of the secretory, endosomal, and lysosomal pathways (Donaldson & Jackson, 2011). Arfs function by recruiting coat proteins onto membranes and through activation of enzymes that alter membrane lipid composition (phospholipase D and phosphatidylinositol 4-phosphate 5-kinase). The five Arf proteins in humans are ubiquitously expressed. Arfs 1, 3, 4, and 5 are mainly observed at the Golgi complex but they also are released into the cytoplasm and have been observed to function in the periphery (Caviston et al., 2014, Kumari and Mayor, 2008). Arf6 is present at the plasma membrane and influences both the cortical actin cytoskeleton and endosomal membrane trafficking (Boshans et al., 2000, Radhakrishna and Donaldson, 1997). Arf6, through its activation of PIP5-kinase, has been observed in clathrin-coated vesicles and has been suggested to facilitate the rapid recycling of the TfR back to the plasma membrane through interaction with the microtubule-motor adaptor protein JNK-interacting protein 4 (JIP4) after the uncoating of clathrin from the vesicle (Montagnac et al., 2011).

Studying the localization and the activation of the Rab and Arf proteins helps in understanding compartment identity and how cargo proteins are sorted and trafficked to their final destinations. In this chapter, we describe a series of experimental approaches to study the roles of Rab and Arf proteins in regulating endosomal membrane traffic. These approaches can assist the investigator who is trying to identify the endosomal compartments that their protein of interest passes through and to understand how that protein is sorted to reach its final destination.