Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Abstract

Clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) co-exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6-associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6-GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6-GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin-coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6-GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.

APEX2‐mediated RAB proximity labeling identifies a role for RAB21 in clathrin‐independent cargo sorting

“…Given our findings that RAB21 and the WASH/retromer complexes are required for “direct CIE cargos” sorting, it could be suggested that these sorting events require F‐actin generation, most probably for enrichment of cargos in tubules as previously reported for ß2AR or for the formation of the tubules. Since loss of RAB21 had a stronger effect on CIE cargo tubule sorting, it is also possible that RAB21 plays a structural role by recruiting other effectors involved in the generation of these tubules, as also observed for RAB22 and RAB35 .…”

Rab35 GTPase: A Central Regulator of Phosphoinositides and F‐actin in Endocytic Recycling and Beyond

“…When clathrin‐mediated endocytic pathway is inhibited, ARF6‐GTP levels are upregulated and functional experiments indicate that Rab35 is on the contrary inactivated. As a consequence, the sorting of cargoes of the ARF6‐dependent pathway is perturbed . At the molecular level, one can imagine that depletion of clathrin inhibits Rab35 activation, as it depends on AP2/clathrin‐binding guanine exchange nucleotide factors (GEFs) .…”

“…This mechanism has been particularly well studied during neurite outgrowth in PC12 cells in response to NGF stimulation (37)(38)(39)(40)(41). Expression of a partially constitutive active mutant (Rab35 Q67L) (42) promotes neurite outgrowth in PC12 cells and in N1E-115 cells, whereas expression of a constitutively active mutant of ARF6 inhibits neurite outgrowth and endocytic recycling (34,38,43,44). Upon nerve growth factor (NGF) stimulation, Rab35 accumulates on ARF6-positive perinuclear recycling endosomes and recruits both MICAL-L1 and ACAP2 (36,38).…”

Escherichia coli K1 utilizes host macropinocytic pathways for invasion of brain microvascular endothelial cells

“…This would suggest that a long‐term block in clathrin‐mediated uptake, as in a siRNA transfection time‐course, reduced an important plasma membrane component for E. coli K1 invasion, diminishing both invasion and signalling. As trafficking through clathrin‐independent endocytic routes has been found to exhibit some dependency on CME, this may explain the apparent effect of CME blocks on E. coli K1 invasion.…”