GTPase networks in membrane traffic

Abstract

Members of the Rab or ARF/Sar branches of the Ras GTPase superfamily regulate almost every step of intracellular membrane traffic. A rapidly growing body of evidence indicates that these GTPases do not act as lone agents but are networked to one another through a variety of mechanisms to coordinate the individual events of one stage of transport and to link together the different stages of an entire transport pathway. These mechanisms include guanine nucleotide exchange factor (GEF) cascades, GTPase-activating protein (GAP) cascades, effectors that bind to multiple GTPases, and positive-feedback loops generated by exchange factor-effector interactions. Together these mechanisms can lead to an ordered series of transitions from one GTPase to the next. As each GTPase recruits a unique set of effectors, these transitions help to define changes in the functionality of the membrane compartments with which they are associated.

Figure 1

Rab-guanine nucleotide exchange factor (GEF) cascade. (a) A Rab GTPase is activated by its own GEF. The GTP-bound active form recruits several effectors to the organelle membrane to fulfill their functions. One of the effectors is a GEF for the downstream Rab: Active Rab A recruits GEF B for the downstream Rab B (left). GEF B activates Rab B, and then active Rab B recruits effectors, including GEF C for the downstream Rab C (middle). Again, GEF C activates the downstream Rab C (right). Thus, several Rab GTPases in the pathway are sequentially recruited to the membrane and efficiently activated. (b) The first Rab GEF cascade was found in yeast. Golgi-localized GTP-bound Rab Ypt31p/Ypt32p interacts with Sec2p, a GEF for the next Rab Sec4p (10).

Figure 2

Cellular localization of GTPase cascades in an epithelial cell. On endosomes, the Rab5-to-Rab7 transition is mediated by the Rab7 guanine nucleotide exchange factor (GEF), SAND1/Mon1-Ccz1 complex, to promote the endocytic pathway (24). The Vps21p-Ypt7p transition (yeast orthologs of Rab5 and Rab7) is also mediated by the Mon1-Ccz1 complex (23). Rab22 and Rab5 also form a cascade on endosomes through the Rab5 GEF, Rabex-5, regulating endosome fusion (28). In the endoplasmic reticulum (ER)-Golgi trafficking pathway, yeast Ypt1p (homologous to Rab1) and Ypt31p/Ypt32p (homologous to Rab11) work sequentially, probably through a Ypt32p GEF (37). Ypt32p interacts with Sec2p, a GEF for the next Rab, Sec4p (Rab8 homolog) (10). Sec2p activates Sec4p on the secretory vesicle, which leads to vesicle tethering and fusion with plasma membrane thorough recruitment of the exocyst complex (11). A mammalian Rab11-Rab8 cascade is mediated by Rabin8, a GEF for Rab8; both Rab8 and Rabin8 are required for primary cilium formation (42, 43). A Rab33b-Rab6 cascade is proposed to work in the intra-Golgi apparatus retrograde trafficking (48). Fission yeast Ryh1p-Ypt3p (mammalian Rab6-Rab11) probably forms a cascade through the Ric1p/Rgp1p complex and works on the exocytic/recycling pathway (49). An Arl3-Arl1 cascade localizes to the Golgi apparatus, where it regulates protein sorting (58, 59). Rab1-Arf1 regulates coat protein I vesicle formation for ER-to-Golgi apparatus transport, which is mediated by GBF1, a Rab GEF for Arf1 (76). On the plasma membrane, Arl4 and Arf6 recruit Arno, a GEF for downstream GTPase Arf1, to regulate cytoskeletal organization and endocytosis (–57). The R-Ras-Arf6-Rac1 pathway is required for cell spreading and migration, which is also mediated by Arno (83, 85, 86). Abbreviations: Arf1–Arf, Rab1–Rab33b, Rac1, members of the GTPase superfamily; R-Ras, a Ras-related protein.

Figure 3

Rab-guanine nucleotide exchange factor (GEF)-effector positive-feedback loop. (a) Upon Rab activation by its GEF, a GTP-bound Rab recruits its effector(s). An effector then binds to the GEF and increases the exchange activity on the Rab GTPase. This Rab-GEF-effector positive-feedback loop stabilizes the active Rab region on the membrane. (b) Rab5 is activated by its GEF Rabex-5, and then active Rab5 recruits its effector Rabaptin-5. Rabaptin-5, in turn, binds to Rabex-5 directly (87, 88). Association with Rabaptin-5 increases Rabex-5 activity toward Rab5, thereby stabilizing Rab5 in a GTP-bound state on the early endosome, which is needed for endosomal fusion (89). (c) In the yeast secretory pathway, Sec2p, which is recruited to the membrane by Ypt32p-GTP, activates Sec4p on the secretory vesicles (12). The GTP-bound form of Sec4p recruits Sec15p, which is a component of the exocyst complex (90). Sec15p binds to Sec2p and displaces Ypt32p (11). Sec2p is able to activate Sec4p, and Sec4p again recruits Sec15p, which leads to vesicle tethering with the plasma membrane through the exocyst complex prior to membrane fusion.