Rab requirements in HOPS-mediated late endosome-lysosome tethering and dissection of fusion factor requirements in Ca²⁺-dependent phagosome-lysosome fusion

Abstract

Through sequential fusion with early- and late endosomes (EEs and LEs) and ultimately lysosomes (LYS), a degradative (endo-)lysosome ((E)LYS is formed. Directionality of fusion is ensured by the action of Ras-like GTPases and their effector proteins, such as tethering proteins. In LE-LYS fusion, tethering is mediated by the HOPS complex and its interaction partners. The chronological order in which these proteins are recruited and act was not established. It is also unclear how HOPS is recruited and anchored to maturing LEs. Ferrofluid (FF)-labelled, purified late endocytic compartments (FFEs) from Arl8b, Arl8a/b, Rab7a, Vps41 (subunit of HOPS), and BORC subunits myrlysin and diaskedin KO cell lines were thoroughly characterised. The examined cells still contained functional (E)LYS. However, transport of endocytosed material to LYS was significantly delayed. Thus, these proteins play a crucial role in transporting cargo to LYS. Kinetic assessment revealed that Rab7a and Vps41 were recruited to maturing endosomes simultaneously and before Arl8, lyspersin (BORC), and LAMP1. Arl8 and BORC subunit levels were reduced on FFEs from Rab7a and Vps41 KOs, indicating that both proteins are recruited first and act before Arl8 and BORC. Vps41 (HOPS) being required before Arl8 raised the question of how HOPS was recruited to LEs. Rab2a was a good candidate for recruiting HOPS, as it binds to the Vps39 subunit of HOPS. A new Rab2a KO HeLa cell line was established. The levels of Vps41 and Vps39 on Rab2a KO FFEs were reduced, as were the levels of other lysosomal proteins (Arl8, mCath D, lyspersin), indicating a role for Rab2a in recruitment of HOPS. However, loss of Rab2a did not displace Vps41 from membranes to the cytosol. Thus, Rab2a cannot be the main membrane anchor of HOPS.<br> In a cell-free fusion assay, requirements of ATP-and cytosol-dependent phagosome-with-LYS fusion (StaFu) can be bypassed by adding micromolar [Ca²⁺] (CaFu). It was unclear whether CaFu required the action of SNARE proteins or NSF (N-ethylmaleimide [NEM] sensitive factor)-dependent priming during the reconstituted fusion reaction. NEM only had moderate inhibitory effects on CaFu, indicating independence of CaFu from NSF-dependent priming during the <em>in vitro</em> fusion reaction. CaFu was moderately inhibited by &alpha;-SNAP^L294A, a potent inhibitor of SNARE-dependent fusion. SNARE participation in CaFu was shown by the inhibitory effect of three preincubated soluble Q-SNAREs. Hence, CaFu depends on the action of SNARE proteins. Further studies must uncover which protein(s) are involved in CaFu and which physiological function this putative shortcut reaction might serve <em>in vivo</em>.