Role of Rab Proteins in Epithelial Membrane Traffic

Sven C.D van IJzendoorn*; Keith E Mostov†; Dick Hoekstra*    * Department of Membrane Cell Biology, University of Groningen, Groningen 9713AV, The Netherlands
† Department of Anatomy, University of California, San Francisco, California 94143, USA

I Introduction

Members of the small GTPase rab protein family play important roles in membrane trafficking. Recent research has provided a vast amount of evidence that rab proteins regulate membrane organization and dynamics. Specific rab proteins are found associated with distinct organelles such as the endoplasmatic reticulum (ER), Golgi apparatus, and endosomes. Most rab proteins are ubiquitously expressed. However, cell types with a highly specialized, e.g., secretory, function often express additional rab proteins, express rab proteins in different ratios, or have adapted the function of specific rab proteins for different needs. Selective expression and/or reorganization of rab protein distribution and functioning may also govern organelle identity.

Epithelial cells are polarized cells that are characterized by the segregation of their plasma membrane (PM) into an apical PM domain facing the lumen and a basolateral PM domain facing the underlying tissue and neighboring cells, each of which displays a distinct protein and lipid composition. Also organelles such as the Golgi apparatus and the endosomal system are distributed in a polarized fashion, and the cytoskeleton displays a highly spatial order in epithelial cells. Such a polarized phenotype allows epithelial cells to perform their delicate function as a barrier between the body and the outside world. Tightly regulated intracellular sorting and trafficking of membranes, and functional tight junctions that prevent the intermixing of apical and basolateral PM components, are required to secure this apical–basolateral polarity (Mostov et al., 2003). A variety of rab proteins, some of which are ubiquitously expressed and some of which appear specifically expressed in epithelia, are known to play a crucial role in epithelial trafficking and, thus, epithelial functioning. These rab proteins are discussed in this review.

II Membrane Traffic in Epithelial Cells

In order to better understand the role of rab proteins in epithelial membrane traffic and polarity, we will first outline the vesicular membrane traffic itineraries for basolateral and apical proteins and lipids in epithelial cells as they are currently understood (Fig. 1).

From the endoplasmatic reticulum (ER), newly synthesized proteins are delivered to the Golgi apparatus in transport vesicles. Following their sequential passage through different Golgi stacks, basolateral and apical PM proteins are sorted at the trans-Golgi network (TGN) and packaged into specific transport carriers for efficient delivery to the respective surface domains (Fig. 1; 1). Transport from the TGN to the PM of some basolateral proteins may involve prior passage through endosomes. The sorting of proteins and lipids is achieved by their segregation into distinct domains within the organelle membrane. Sorting of basolateral proteins is mediated by well-described sorting signals encoded in their cytoplasmic domains that typically include tyrosine, dileucine, and monoleucine motives and clusters of acidic amino acids. Basolateral sorting signals are recognized by cytosolic proteins, including the μ1b adaptin subunit (Fölsch et al., 1999; Gan et al., 2002; Simmen et al., 2002, Mostov et al., 2000). μ1b adaptin is part of the AP-1 adaptor complex that also binds to clathrin, in this way causing basolateral proteins to become concentrated in clathrin-coated vesicles (Hirst and Robinson, 1998). Sorting of apical proteins is less understood but may be governed by carbohydrate (N-glycan, O-glycan) modifications in the ectodomain (Scheiffele et al., 1995; Scheiffele and Fullekrug, 2000) and is generally thought to involve their association with (glyco)sphingolipid and cholesterol-enriched microdomains called rafts, either directly [e.g., glucosylphosphatidylinositol (GPI)-anchored proteins] or indirectly via binding to other raft proteins such as lectins (Simons and Ikonen, 1997; Maier et al., 2001, Aït Slimane and Hoekstra, 2002). It should be noted, however, that some nonraft-associated proteins are sorted to the apical surface via less understood cytoplasmic apical-sorting signals (Gokay et al., 2001; Takeda et al., 2003; Jacob and Naim, 2001). Different apical-sorting mechanisms possibly include the segregation of apical cargo into distinct apical PM-targeted vesicular carriers (Jacob and Naim, 2001).

There are also examples of raft-associated proteins traveling to the basolateral surface (Sarnataro et al., 2002; Aït Slimane et al., 2003), indicating that raft association per se is thus not sufficient for apical targeting. Moreover, nonpolarized fibroblasts also sort apical and basolateral proteins, which are either delivered to the “uniform” PM (Yoshimori et al., 1996; Keller et al., 2001; Tuma et al., 2002) or selectively retained intracellularly. Interestingly, protein glycosylation, which mediates apical delivery in epithelial cells, can provide a signal for surface transport in nonpolarized fibroblasts (Scheiffele and Fullekrug, 2000). Upon epithelial polarization, basolateral and apical proteins are selectively targeted to the lateral and apical PM domain, respectively, as demonstrated by advanced confocal and time-lapse internal reflection fluorescence microscopy in living cells (Kreitzer et al., 2003). The molecular mechanisms that govern targeting following sorting are largely obscure, but, at least for apical proteins, appear to involve the concerted action of microtubules and the asymmetric distribution of docking and membrane fusion machineries, including syntaxin 3 (Kreitzer et al., 2003). The multiprotein exocyst complex appears to mediate the targeting of basolateral proteins to the tight junction area (Lipschutz and Mostov, 2002) and also interacts with microtubules (Vega and Hsu, 2001). Where at the cell surface apical or basolateral protein-containing vesicles are eventually targeted in polarized epithelia is subject to cell type (e.g., hepatocytes target many apical proteins to the basolateral surface prior to apical delivery) and differentiation state of the cells (Zurzolo et al., 1992; van Adelsberg et al., 1994). In addition, extracellular cues such as interaction between epithelial cells or interaction with extracellular matrix (ECM) components and, subsequently, rearrangements of the microtubule and actin network play an important role in defining sites at the cell surface for vesicle targeting (van Adelsberg et al., 1994; Yeaman and Nelson, 1997).

The large variety of transport vesicles with different lipid and protein composition known to exit from the Golgi/TGN implies a highly organized and dynamic membrane architecture/organization of this organelle. Factors that affect organelle membrane curvature, e.g., the formation of tubular or globular extensions, can facilitate or frustrate the interaction of those membranes with cytosolic proteins (e.g., clathrin) required to form specific transport vesicles (Cluett et al., 1993). Furthermore, cytoplasmic phospholipase A2 (PLA2) activity contributes to the formation of membrane tubules from the Golgi and endosomes, and PLA2-induced endosomal tubules have been reported to be involved in the recycling of the transferrin receptor to the PM (Drecktrah and Brown, 1999; de Figuieredo et al., 2001). Thus, although polarized sorting of proteins and lipids is mediated by their recruitment into membrane domains, the nature and presumed plasticity of such domains and their interaction with other intracellular components that govern their fate remain largely obscure.

Upon arrival at the cell surface, many proteins and lipids are internalized via a process called endocytosis (Fig. 1; 2). Endocytosis comprises a variety of distinct ways by which molecules can enter the cell, including clathrin- and raft-mediated routes and pinocytosis (Mukherjee et al., 1997). Apical endocytic activity is generally lower than that of...