Luis S. Mayorga

Membrane trafficking along the phagocytic pathway

Phagosome maturation involves extensive remodelling of the phagosomal membrane as a result of intracellular transport events. Newly formed phagosomes exchange membrane-associated and soluble proteins with early endosomes by fusion. Budding of vesicles from the phagosome and fusion with Golgi-derived vesicles may also contribute to the remodelling of the phagosomal compartment. As a consequence of changes in membrane composition, phagosomes acquire the ability to fuse with late endocytic compartments. In vitro reconstitution and other studies suggest that the trafficking events underlying phagosome maturation require several GTP-binding proteins, including Rab5 and Galphas, NSF-SNAP-SNARE complexes and coatomers.

Regulatory role for GTP-binding proteins in endocytosis

Guanosine 5-triphosphate (GTP)-binding proteins have been implicated in the transport of newly synthesized proteins along the secretory pathway of yeast and mammalian cells. Early vesicle fusion events that follow receptor-mediated endocytosis as measured by three in vitro assays were blocked by guanosine 5-O-(3-thiotriphosphate) and aluminum fluoride. The effect was specific for guanosine nucleotides and depended on the presence of cytosolic factors. Thus, GTP-binding proteins may also have a role in the transport of molecules along the endocytic pathway.

Inhibition of endocytic transport by aluminum fluoride implicates GTPases as regulators of endocytosis.

It is now well established that GTP-binding proteins are important regulators of vesicular transport. Recent work has shown that multiple GTPases (both monomeric and heterotrimeric) are required for trafficking. In the present study we have used aluminum fluoride (AIF), a reagent that activates trimeric G proteins, as a tool to study the involvement of this family of GTPases in the regulation of endocytosis in intact cells. Our results indicate that AIF inhibits fusion of early endosomes with an intracellular proteolytic compartment. Using the mixing of sequentially internalized ligands as a measure of endocytosis, we found that AIF inhibited endocytic transport as assessed by both biochemical and morphological methods. Taken together these results suggest that AIF affects membrane fusion, a common step in vesicular transport. To further examine the effects of AIF we tested this compound in a cell-free assay that reconstitutes fusion among endosomes. AIF affected endosomal fusion in a different way than did GTP gamma S, an agent that activates both trimeric and small GTPases. Our results suggest that the coordinated activation of both classes of GTPases are required for efficient endocytic transport.

Reconstitution of intracellular vesicle fusion in a cell-free system after receptor-mediated endocytosis.

Publisher Summary This chapter describes fusion assay that based on two proteins: mannosylated monoclonal immunoglobulin G (Man-IgG) specific for dinitrophenol (DNP) groups, and DNP-derivatized β-glucuronidase. These two molecules are efficiently internalized by cell lines that express the macrophage mannose receptor and form a stable immune complex when they are localized in the same compartment. This complex can be quantified using a fluorescent substrate for β-glucuronidase. The assay is very sensitive, reproducible, and relatively inexpensive. The chapter develops two probes that have proved suitable for these studies because both are efficiently internalized by receptor-mediated endocytosis and, when present in the same compartment, form a complex that can be easily quantified by enzymatic analysis. These probes may potentially be used to study other fusion steps along the intracellular route followed by endocytosed ligands. Reconstitution of these events in cell-free systems will help clarify the role of fusion in the endocytic pathway. In vitro assays may also be applied to the study of the molecular mechanisms involved in the specific recognition between fusogenic vesicles and membrane fusion.

GTPases: Key regulatory components of the endocytic pathway

GTP-binding proteins or GTPases are versatile cyclic molecular switches (Gilman, A.G. 1987 ; Bourne et al., 1990, 1992). In the past few years there has been an explosion of interest in unraveling the role of GTPases in membrane traffic (Balch et al., 1990; Bourne, H.R., 1988). GTP-binding proteins are classified into two broad families: ras-like monomeric GTP-binding proteins and the heterotrimeric G proteins. Evidences from genetics, immunolocalization, and functional assays has established that two subfamilies of monomeric GTP-binding proteins, the Rab and ARF subfamilies, are required for membrane traffic (Pfeffer, S.R., 1992; Rothman, J.E. and Orci, L., 1992). More recently, attention has turned to the role of heterotrimeric G proteins in membrane traffic (Balch et al., 1992; Barr et al., 1992). This review will focus solely on the recent data that suggest that heterotrimeric G proteins and monomeric GTP-binding proteins are involved in endosome-endosome fusion and in endocytic transport.

Identification of Proteins Involved in Endosome Fusion: Implications for Toxin Activity

Endocytosis of proteins is a fundamental aspect of protein transport required for cell survival. Binding of a number of ligands including hormones, growth factors, nutrients and antigens to cell-surface receptors initiates internalization of these macromolecules. The receptor-ligand complexes that form at the cell surface sequester within clathrin-coated pits and the pits pinch off the plasma membrane to form coated vesicles1. After uncoating, the vesicles fuse with other endocytic vesicles, the compartment acidifies and the ligands dissociate from their receptors2. The lumenal contents of the endocytic vesicles are commonly delivered to lysosomes or the Golgi apparatus while the majority of the membrane components are recycled to the plasma membrane3.