Structure, Exchange Determinants, and Family-Wide Rab Specificity of the Tandem Helical Bundle and Vps9 Domains of Rabex-5

Abstract

The Rab5 GTPase, an essential regulator of endocytosis and endosome biogenesis, is activated by guanine-nucleotide exchange factors (GEFs) that contain a Vps9 domain. Here, we show that the catalytic core of the Rab GEF Rabex-5 has a tandem architecture consisting of a Vps9 domain stabilized by an indispensable helical bundle. A family-wide analysis of Rab specificity demonstrates high selectivity for Rab5 subfamily GTPases. Conserved exchange determinants map to a common surface of the Vps9 domain, which recognizes invariant aromatic residues in the switch regions of Rab GTPases and selects for the Rab5 subfamily by requiring a small nonacidic residue preceding a critical phenylalanine in the switch I region. These and other observations reveal unexpected similarity with the Arf exchange site in the Sec7 domain.

Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

“…The intrinsic nucleotide exchange rate, as reflected by the GTP␥S loading rate, was extremely low in the presence of 5 mM Mg 2ϩ and served as a negative control in these experiments ( Figure 2A). The GEF domain alone (residues 135-399) strongly stimulated the GTP␥S loading of Rab5-GDP (Figure 2A), consistent with a previous report (Delprato et al, 2004). In this regard, full-length Rabex-5 had little exchange activity (Esters et al, 2001;Lippe et al, 2001).…”

“…In addition, the core GEF domain (residues 132-391) was also highly active (k cat Ͼ0.1 s Ϫ1 ) (Delprato et al, 2004). These observations might reflect an "autoinhibition" mechanism in which other domain(s) in Rabex-5 blocks the GEF domain activity, and Rabaptin-5 interaction may relieve this inhibition.…”

“…Rabex-5 itself shows very little GEF activity in vitro, and it requires interaction with Rabaptin-5 to gain activity (Esters et al, 2001;Lippe et al, 2001). Furthermore, the core GEF domain (Vps9 domain plus the upstream helical bundle and the downstream ␣-helix, residues 132-391) of Rabex-5 is also much more active than the full-length protein in in vitro biochemical assays (Delprato et al, 2004). It is unclear why the full-length Rabex-5 has such low GEF activity in vitro and how interaction with Rabaptin-5 can stimulate this activity.…”

“…Rabex-5 contains multiple functional domains (Delprato et al, 2004). A coiled-coil domain downstream of the GEF domain may mediate the binding to Rabaptin-5, as shown in a yeast two-hybrid assay , whereas a Zn 2ϩ finger (ZnF) domain at the N terminus is shown to bind ubiquitin (Lee et al, 2006;Mattera et al, 2006;Penengo et al, 2006).…”

GD1b-Derived Gangliosides Modulate FcεRI Endocytosis in Mast Cells

“…The AP2 adaptor complexes, consisting of four adaptins (Boehm and Bonifacino 2001;Pearse 1975), associate with the receptor and recruit clathrin to the plasma membrane, leading to the formation of clathrin-coated vesicles (Bonifacino and Traub 2003), which are responsible for the initial transport of receptors to primary endosomes (Marsh and McMahon 1999). After losing their clathrin coat, the early endosomes express the GTPase Rab5 (Delprato et al 2004;Rios et al 2008), syntaxin 1/2 (Bonifacino and Hurley 2008;Carlton et al 2004;Carlton et al 2005;Rojas et al 2008), EEA1 (Simonsen et al 1998), and Rab4 ( Van der Sluijs et al 1991), among others. The early endosomes have the primary function of sorting internalized cargo to different cellular compartments (Dunn et al 1989;Mayor et al 1993).…”