Identification and characterisation of novel Mss4-binding Rab GTPases

Abstract

The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.

Stratum, a Homolog of the Human GEF Mss4, Partnered with Rab8, Controls the Basal Restriction of Basement Membrane Proteins in Epithelial Cells

“…Altogether, these results suggest that Strat acts as a GEF for Rab8 during the basal restriction of BM deposition. This conclusion is supported by the data that Rab8a interacts with Mss4 in mammalian cells and has weak GEF activity for Rab8a in vitro (Itzen et al, 2006; Wixler et al, 2011). We also showed that Strat phenotype is rescued by hMss4, suggesting that Strat and hMss4 share similar activities.…”

“…In Drosophila , Rab8 and Rab10 are paralogs, sharing an amino acid sequence identity of 67%. In addition, mammalian Mss4/RabIF can act as a GEF for Rab8a (Itzen et al, 2006; Wixler et al, 2011). First, we examined the effects of Rab8 on BM proteins.…”

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

“…Rabin8 and GRAB are specific GEFs for Rab8, in contrast to the rather promiscuous protein MSS4, which shows GEF activity toward Rab1, Rab3, Rab10, and Rab13 in addition to Rab8 (25,39,40). MSS4 is a catalytically inefficient GEF with a k cat /K m value about four times lower than that of Rabin8/ GRAB.…”

Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport

“…These results led to the conclusion that GEFs are important and essential factors for the targeting of Rabs to specific membranes or membrane compartments. This mechanism can only apply if RabGEFs are localized to membranes, and this appears to be the case for a large number of RabGEFs , possibly for all of them except for Mss4, which is probably not a bona fide RabGEF even if it shares some properties with GEFs (Itzen et al, 2006;Wixler et al, 2011).…”