Membrane identity and GTPase cascades regulated by toggle and cut‐out switches

Abstract

Key cellular functions and developmental processes rely on cascades of GTPases. GTPases of the Rab family provide a molecular ID code to the generation, maintenance and transport of intracellular compartments. Here, we addressed the molecular design principles of endocytosis by focusing on the conversion of early endosomes into late endosomes, which entails replacement of Rab5 by Rab7. We modelled this process as a cascade of functional modules of interacting Rab GTPases. We demonstrate that intermodule interactions share similarities with the toggle switch described for the cell cycle. However, Rab5‐to‐Rab7 conversion is rather based on a newly characterized ‘cut‐out switch’ analogous to an electrical safety‐breaker. Both designs require cooperativity of auto‐activation loops when coupled to a large pool of cytoplasmic proteins. Live cell imaging and endosome tracking provide experimental support to the cut‐out switch in cargo progression and conversion of endosome identity along the degradative pathway. We propose that, by reconciling module performance with progression of activity, the cut‐out switch design could underlie the integration of modules in regulatory cascades from a broad range of biological processes. The transition from early to late endosomes is regulated by the loss of the small GTPase Rab5 and the concomitant acquisition of Rab7 in a mechanism termed Rab conversion ([Rink et al , 2005][1]). The behaviour of the two master GTPases creates a paradox: on the one hand, early endosomes are required to maintain Rab5 and increase Rab5's activity as they (1) receive incoming cargo from the plasma membrane, (2) grow in size through homotypic early endosome fusion and (3) package cargo destined for degradation while sorting recycling cargo to the cell surface. On the other hand, when cargo has sufficiently accumulated in fewer and larger endosomes and the surface density of Rab5 reaches its peak, Rab5 needs to be switched off and substituted by Rab7 to irreversibly commit cargo for degradation. To resolve this paradox, we considered these two master GTPases as modules and applied a combination of theoretical and experimental approaches to unravel the yet unknown design principles of the switch system. Here, we identified two design principles that can explain the maintenance and dynamic transition between successive Rab domains or Rab modules, each requiring only a single inhibitory interaction ([Figure 2][2]): (1) Toggle switch. Rab5 displays cooperative auto‐activation and suppresses Rab7. (2) Cut‐out switch. Rab5 activates Rab7; Rab7 displays cooperative auto‐activation and suppresses Rab5. According to model 1, Rab5 auto‐activates and controls the level of Rab7 by a negative feedback loop. To perform such a task, it is necessary for Rab5 to maintain its level above a threshold; therefore, by decreasing the level of Rab5, Rab5 will be replaced by Rab7. According to model 2, Rab5 activates Rab7 until Rab7 reaches a threshold upon which it inactivates Rab5 through a negative feedback loop and hence Rab5 activity needs to increase for Rab7 activity to pass its threshold. So far, model 2 is best supported by the experimental data ([Figure 3][3] and [Table I][4]). Therefore, we propose that Rab conversion is operated by a cut‐out switch analogous to an electrical safety‐breaker ([Morecoft and Hehre, 1933][5]; [Oliver, 1990][6]) controlled by Rab7. To our knowledge, this is the first example of a cut‐out switch used in a biological system. We propose that the design principle shown here is not limited to Rab conversion but may underlie other modules posing a similar paradox. This proposal is supported by a series of experimental evidences for the necessary components of predicted cut‐out switches in other modules centred on Rab as well as other GTPases in intracellular transport. In conclusion, the description of the Rab5–Rab7 system as small functional units, or modules, represented by individual Rab domains ([Miaczynska and Zerial, 2002][7]) gives the possibility to further develop a more comprehensive model of the entire endocytic pathway, taking into account the recycling branch as well as further molecular components of the endocytic machinery, for example, coats and SNARE proteins ([Heinrich and Rapoport, 2005][8]). It is conceivable that the cut‐out switch described here could be a design principle shared by other regulatory cascades from a broad range of biological processes. Mol Syst Biol. 4: 206 [1]: #ref-33 [2]: #F2 [3]: #F3 [4]: #T1 [5]: #ref-23 [6]: #ref-24 [7]: #ref-22 [8]: #ref-10