The PripA-TbcrA complex-centered Rab GAP cascade facilitates macropinosome maturation in Dictyostelium

Abstract

Macropinocytosis, an evolutionarily conserved mechanism mediating nonspecific bulk uptake of extracellular fluid, has been ascribed diverse functions. How nascent macropinosomes mature after internalization remains largely unknown. By searching for proteins that localize on macropinosomes during the Rab5-to-Rab7 transition stage in Dictyostelium, we uncover a complex composed of two proteins, which we name PripA and TbcrA. We show that the Rab5-to-Rab7 conversion involves fusion of Rab5-marked early macropinosomes with Rab7-marked late macropinosomes. PripA links the two membrane compartments by interacting with PI(3,4)P2 and Rab7. In addition, PripA recruits TbcrA, which acts as a GAP, to turn off Rab5. Thus, the conversion to Rab7 is linked to inactivation of the upstream Rab5. Consistently, disruption of either pripA or tbcrA impairs Rab5 inactivation and macropinocytic cargo processing. Therefore, the PripA-TbcrA complex is the central component of a Rab GAP cascade that facilitates programmed Rab switch and efficient cargo trafficking during macropinosome maturation.

PIKfyve is required for phagosomal Rab7 acquisition and the delivery and fusion of early macropinosomes to phagosomes

“…During classical endocytosis, Rab5 accumulates at early stages and exchanges with Rab7 as the vesicles mature (Rink et al, 2005). A similar Rab5-Rab7 exchange has been described during both phagosome and macropinosome maturation at around the same time we observe PIKfyve-GFP recruitment (Kerr et al, 2006; Poteryaev et al, 2010; Tu et al, 2022; Vieira et al, 2003).…”

“…Co-expression of RFP-Rab5a and GFP-Rab7A allowed us to follow the dynamics of both proteins to newly formed phagosomes in wildtype and ΔPIKfyve cells ( Figure 2A and B, Movie 3 ). In wild-type cells, as previously reported (Tu et al, 2022), RFP-Rab5A was highly enriched on perinuclear endosomes, and to a lesser extent at the plasma membrane. We also observed that RFP-Rab5A became slightly enriched at phagocytic cups just prior to closure.…”

“…As loss of PI(3,5)P2/Rab7 delivery would explain the defective Rab7 accumulation and maturation observed in ΔPIKfyve cells, we sought to identify the origin of these PI(3,5)P2/Rab7 positive vesicles. We observed above that early macropinosomes are marked by both these components (Figure 5D-E), and others have shown that macropinosomes undergo homotypic fusion in both Dictyostelium and mammalian cells (Dolat and Spiliotis, 2016;Hamasaki et al, 2004;Neuhaus et al, 2002;Tu et al, 2022). Using sequential pulses of different coloured dextrans, we were also able to observe macropinosomes fusing with one another from relatively early stages of maturation (Figure S6B).…”

“…2xFYVE-GFP (pJSK418) uses the sequence from the human Hrs gene and was previously described (Calvo-Garrido et al, 2014). A GFP-Rab7A (pTH70) plasmid was kindly gifted by Huaquing Cai (Tu et al, 2022). Co-expression plasmids were created by first cloning the RFP-fusion sequence into the pDM series shuttle vectors (pDM1042 and pDM1121), before cloning as an NgoMIV fragment into the appropriate GFP-expression vector.…”

The GxcM-Fbp17/RacC-WASP signaling cascade regulates polarized cortex assembly in migrating cells

“…To identify proteins that interact specifically with GxcM, immunoprecipitation experiment and mass spectrometry analysis were carried out as described previously with minor modification (Tu et al, 2022). Briefly, cells expressing GxcM-GFP, GxcM N790- GFP, or Teep1-GFP were starved in DB for 3 h, lysed in ice-cold lysis buffer (10 mM Hepes pH 7.2, 100 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM NaF, 0.5 mM Na 3 VO 4 , complete EDTA-free protease inhibitor (Roche), 1 mM DTT) with the addition of 15 mM EDTA, and incubated for 5 min on ice.…”