Coordinated protein sorting, targeting and distribution in polarized cells

Abstract

The polarized distribution of functions in polarized cells requires the coordinated interaction of three machineries that modify the basic mechanisms of intracellular protein trafficking and distribution. First, intrinsic protein-sorting signals and cellular decoding machineries regulate protein trafficking to plasma membrane domains; second, intracellular signalling complexes define the plasma membrane domains to which proteins are delivered; and third, proteins that are involved in cell-cell and cell-substrate adhesion orientate the three-dimensional distribution of intracellular signalling complexes and, accordingly, the direction of membrane traffic. The integration of these mechanisms into a complex and dynamic network is crucial for normal tissue function and is often defective in disease states.

Figure 1. A generic post-translational pathway for protein trafficking to the plasma membrane

After synthesis in the endoplasmic reticulum (ER), membrane proteins are sorted into vesicles by the coatomer protein complex-II (COPII) machinery and delivered to the Golgi complex by vesicle-tethering and SNARE machineries. Intra-Golgi transport and retrograde transport from the Golgi to the ER are regulated by the COPI machinery. At the trans-Golgi network (TGN), proteins are sorted into vesicles by intrinsic sorting motifs and cytoplasmic adaptor complexes, and are transported along cytoskeletal elements to the plasma membrane. Protein delivery to the plasma membrane is mediated by vesicle-tethering and SNARE machineries. Some proteins (for example, ligand–receptor complexes) are internalized through another set of adaptors and delivered to an endosome (E), from which they might be recycled back to the plasma membrane.

Figure 2. Superimposing protein sorting demands on the generic trafficking pathway

In polarized cells, such as epithelia and neurons, protein processing occurs along the generic pathway between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN; see also FIG. 1). However, proteins in the TGN might be sorted into various different vesicles through the recognition of different intrinsic sorting motifs (TABLE 1) and cytoplasmic adaptor complexes. These vesicles are then targeted either directly or indirectly, through an endosome (E), to different plasma membrane domains (designated as domains I and II) along cytoskeletal elements. These cytoskeletal elements might have different orientations (or polarity) relative to the different membrane domains. Vesicle delivery to each plasma membrane domain is mediated by different vesicle-tethering and SNARE complexes. Some proteins are internalized through another set of adaptors and delivered to an endosome, from which they might be recycled back to the original plasma membrane domain, or to the other domain by trancytosis, depending on the presence (or activation or inactivation) of specific protein-sorting motifs. AP, adaptor protein; CL, clathrin; COP, coatomer protein complex.

Figure 3. Signalling complexes and scaffolds on the cytosolic face of the membrane define and stabilize membrane domains

a | Three signalling complexes (Crumbs, partitioning defective (PAR) and Scribble) associate with the cytoplasmic surface of the plasma membrane around sites of cell adhesion, which demarcates different plasma membrane domains — in this example, the apical membrane (top) and the basolateral membrane (bottom). Crumbs protein (CRB) is a transmembrane protein, but mechanisms of binding of the PAR and Scribble complexes are poorly understood (see the main text). Proteins within each of these three complexes physically interact, as do PATJ (PALS1 (protein associated with LIN-7)-1-associated tight-junction protein) and PAR6 in the Crumbs and PAR complexes. Atypical protein kinase C (aPKC; in the PAR complex) genetically interacts with, and stabilizes, CRB (broken line, +), and phosphorylates and negatively regulates (broken line, −) lethal giant larvae (LGL) in the Scribble complex. Overall, the PAR complexes reinforce the localization and activity of the Crumbs complex (thick arrow), and the PAR and Scribble complexes mutually antagonize each other (inhibition lines). b | PAR3 phosphorylation by PAR1 results in the binding of phosphorylated PAR3 and PAR5, and dislocation of PAR3 from the membrane into the cytoplasm. Similarly, PAR1 phosphorylation by aPKC or PAR4 results in the binding of phosphorylated PAR1 and PAR5, and dislocation of PAR1 into the cytoplasm (see the main text for details). SCRB, Scribble protein; STD, stardust.

Figure 4. Roles of signalling complexes on the cytosolic face of the membrane control phosphoinositide distribution and vesicle trafficking

a | The partitioning defective (PAR) complex structurally and functionally interacts with Rho family GTPases, either by direct binding of PAR6 to active CDC42 (CDC42–GTP) or regulation of the guanine nucleotide-exchange factor T-cell-lymphoma invasion and metastasis-1 (TIAM1), which locally activates RAC1 (forming RAC1–GTP). These GTPases locally regulate actin organization. The PAR complex and associated CDC42 and RAC1 also locally regulate phospholipid synthesis through the direct binding of PAR3 to phosphatase and tensin homologue (PTEN), which generates phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P₂), and through activation of phosphoinositide 3-kinase (PI3K), which generates phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃). PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ might be localized to different domains of the plasma membrane — in this example, the apical membrane (top) and the basolateral membrane (bottom). b | The Scribble complex locally regulates actin organization by Scribble protein (SCRB) binding to the PAK-interacting exchange-factor-β–G protein-coupled receptor kinase-interactor (βPIX–GIT) complex, which locally regulates CDC4 and RAC1 in activating the actin-polymerization machinery (Wiskott–Aldrich syndrome protein (WASP) or WASP family verprolin-homologous protein (WAVE), and actin-related protein-2/3 (ARP2/3)). Local activity of the βPIX–GIT complex and actin polymerization affect the organization of the exocyst vesicle-tethering complex and vesicle delivery to the plasma membrane.

Figure 5. Polarized cells form from the hierarchical integration of three fundamental mechanisms

Cell adhesion to the extracellular matrix (ECM) or to adjacent cells provides an extrinsic spatial cue (dark blue) that signals the assembly of intracellular scaffolds (light blue): Crumbs and PAR3 specify the apical domain and Scribble specifies the basolateral domain. Intracellular scaffolds define distinct plasma membrane (PM) domains and separate them by inserting a molecular fence that acts as a diffusion barrier. Finally, protein-sorting codes and cellular decoding machineries distinguish membrane components destined for distinct plasma membrane domains, sorting them into distinct vesicular transporters at the level of the trans-Golgi network (TGN) and endosomes. The transport vesicles are equipped to recognize either domain I or II, thus constructing the biochemical heterogeneity that is characteristic of the polarized cell surface (grey). The integration of these mechanisms is important for normal tissue function and is often defective in disease states.