Rabaptin-5alpha/rabaptin-4 serves as a linker between rab4 and gamma1-adaptin in membrane recycling from endosomes

Abstract

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5a and its putative partner g 1 -adaptin in membrane recycling. We found that rabaptin-5a forms a ternary complex with the g 1 ±s 1 subcomplex of AP-1, via a direct interaction with the g 1 -subunit. The binding site for g 1 -adaptin is in the hinge region of rabaptin-5a, which is distinct from rab4-and rab5-binding domains. Endogenous or ectopically expressed g 1 -adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5a and g 1 -adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5a caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5a± g 1 -adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5a and g 1 -adaptin regulate membrane recycling.

In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

“…Initially, rabaptin-5 was identified as an interactor of rab5 (Stenmark et al, 1995) and rabex-5 as a rab5 guanine nucleotide exchange factor (Horiuchi et al, 1997). Rabaptin-5 in addition was shown to bind to rab4 and AP-1 (Vitale et al, 1998;de Renzis et al, 2002;Deneka et al, 2003). On immunodepletion of rabaptin-5/rabex-5 from the cytosol with an anti-rabaptin-5 antibody ( Figure 8A), formation of endosomal vesicles was reproducibly stimulated (Figure 8B), suggesting an inhibitory role of the complex.…”

“…Three rab proteins have been implicated in regulating receptor recycling. Rab4 was implicated in regulating recycling from endosomes to the cell surface in nonpolarized and to the apical surface in polarized cells (van der Sluijs et al, 1992;Mohrmann et al, 2002;Deneka et al, 2003).…”

“…Interestingly, rabaptin-5 had been shown to interact also with the ␥ ear domains of AP-1 and GGAs, a family of ARF1-dependent clathrin adaptors (Hirst et al, 2000;Shiba et al, 2002;Deneka et al, 2003;Mattera et al, 2003). A possible role of rab4 might thus be to recruit rabaptin-5/rabex-5 to recycling endosomes as a docking site for AP-1 adaptors.…”

“…In addition, brefeldin A (BFA), an inhibitor of guanine nucleotide exchange factors of the GTPase ADPribosylation factor 1 (ARF1) involved in recruiting AP-1-and AP-3-containing coats, partially inhibited transferrin receptor recycling in nonpolarized cells . Overexpression of rabaptin-5, an interactor of the ␥-subunit of AP-1 (Hirst et al, 2000;Shiba et al, 2002) as well as of rab4 and rab5 (Vitale et al, 1998), was shown to inhibit transferrin receptor recycling, unless ␥-adaptin was overexpressed simultaneously (Deneka et al, 2003).…”

“…In addition, brefeldin A (BFA), an inhibitor of guanine nucleotide exchange factors of the GTPase ADPribosylation factor 1 (ARF1) involved in recruiting AP-1-and AP-3-containing coats, partially inhibited transferrin receptor recycling in nonpolarized cells . Overexpression of rabaptin-5, an interactor of the ␥-subunit of AP-1 (Hirst et al, 2000;Shiba et al, 2002) as well as of rab4 and rab5 (Vitale et al, 1998), was shown to inhibit transferrin receptor recycling, unless ␥-adaptin was overexpressed simultaneously (Deneka et al, 2003).AP-1/clathrin coats on endosomes may, however, be involved in other functions than recycling to the plasma membrane (Hinners and Tooze, 2003) is transported via endosomes to the Golgi, was found to be colocalized in recycling endosomes with AP-1 and clathrin and accumulated in transferrin receptor-positive endosomes upon BFA treatment (Mallard et al, 1998). In cells lacking the 1A subunit of AP-1, the mannose-6-phosphate receptor was found to accumulate in endosomes, rather than in the TGN, supporting an AP-1-dependent pathway from endosomes to the TGN (Meyer et al, 2000).…”