Endosomal sorting of VAMP2, -3 and -4 with Rab GTPases

Endosomal sorting and trafficking are facilitated by Rab GTPases and SNAREs. These groups of proteins are important for endosomal identity and progression in the endocytic pathway due to their fusogenic properties. Although the function and localisation of SNARE complexes have been systematically studied in neurons, very few live cell studies of VAMPs in the endocytic pathway are available. Here, we want to characterise the R-SNAREs; VAMP2, VAMP3 and VAMP4 in live HeLa K cells and analyse the endosomal pathway, to understand more about their function during endosomal progression. In this study we found VAMP2, -3 and -4 localised to early endosomes, where VAMP3 was additionally present on late endosomes. Dynamically, FRAP experiments could show us that VAMP2, -3 and -4 is constantly exchanged on the early endosomal membrane. Tracking VAMP2, -3 and -4 through early endosomal maturation we found the formation of microdomains, and convergence finally led to a detachment of a newly formed endosome positive for VAMP2, -3 and -4 together with Rab5 or Rab22a. Manipulating the expression level of Rab5 or Rab22a changed the maturation and specifically changed the sorting of VAMP3. The maturation and the detachment of the respective VAMPs were found to be specifically regulated by Rab5 and Rab22a. We show here for the first time that VAMP2, -3 and -4 as integrated endosomal proteins are sorted to nascent endosomes. Sorting of VAMPs together with important GTPases during endosomal maturation provides a functional platform for the newly formed early endosome. With this we show that sorting and recycling of membrane associated and integrated proteins is a generic mechanism for maintaining a functional endosomal pool.