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Not much is known about GMAP-210 interactors. TheS.cerevisiaehomologue of GMAP- 210, Rud3p, is shown to bind to the Golgi apparatus via its interaction with the small GTPase Arf1p (Gillinghamet al., 2004).

3.4.1 Binding Of The GRAB Domain To Arf1 Is Regulated

The whole family of small ADP-ribosylation factor and ribosylation factor-like proteins was tested for interaction in yeast 2-hybrid experiments (Fig.13, p.37). Full-length GMAP- 210 interacts with the small GTPases Arl4A, B and C and Arl16. Arl16 does not interact with the GMAP-210 GRAB domain, but Arl4A, B and C do and the interaction is abol- ished by the mutation of the two essential residues aspartate and leucine in the GRAB domain (Fig.14, p.38). However, further studies reveal, that the Arl4 family does not localise to the Golgi apparatus, but is found on the plasma membrane (Fig.16, p.40)

(Hofmann et al., 2007). Arl4 recruits the guanine nucleotide exchange factor cytohesin and thus activates Arf6. Pulldown experiments with recombinant Arl4 (Fig.18, p.43) and immunoprecipitation (Fig.19, p.44) show no binding of GMAP-210 and Arl4. Surprisingly, the active mutant of Arf1 interacts in yeast 2-hybrid with a fragment, which contains only the GMAP-210 GRAB domain and mutation of the GRAB domain abolishes this. Arf1 does, however, not interact with full length GMAP-210 (Fig.12, p.35). Yeast 2-hybrid mapping of GMAP-210 shows, that Arf1 interaction is inhibited by a part N-terminally from the GRAB domain (Fig.22, p.47). This hints at regulation of Arf1 binding to the GMAP-210 GRAB domain in full-length GMAP-210. The GRAB domain is activated for Arf1 binding upon interaction with a yet unidentified factor.

3.4.2 GMAP-210 Interacts With The Small GTPase Rab1

Rab GTPases are involved in all transport steps and give identity to membrane compart- ments (Pfeffer, 2001; Zerial and McBride, 2001; Short et al., 2005). They are essential components of intracellular trafficking. GMAP-210 was screened against a yeast 2-hybrid library of the complete Rab GTPase family (Fig.30, p.58). Several Rab GTPases show interaction with GMAP-210, including Rab1 and Rab2. Rab1 co-localises with GMAP- 210 on the cis-Golgi of HeLa L cells (Fig.31, p.59), Rab2 is clearly localised on a different compartment, the medial Golgi (Shortet al., 2001). Pulldown experiments with all pos- itive Rab GTPases found in the yeast 2-hybrid screen (Fig.33, 34, p.61, 62) confirm, that Rab1 interacts with GMAP-210 in a nucleotide dependent manner. Yeast 2-Hybrid experiments were further used to map down the interaction domain for Rab1 on GMAP- 210 (Fig.32, p.59). Amino acids 855 to 1332 can bind Rab1. Interaction with Rab2 is stronger, but due to the localisation of Rab2 and its inability to pull down GMAP-210, this is likely of no importance. Even more interesting is the fact, that GRAB mutant GMAP-210 is recruited to the Golgi of HeLa L cells, when endogenous GMAP-210 is depleted by siRNA. This targeting capability also maps to the same fragment as Rab1 binding (Fig.27, p.53). It is thus possible, that GMAP-210 binds to Rab1, but only in combination with GRAB domain targeting (Fig.68). Without the GRAB domain bind- ing signal, the Rab1 interaction domain is too weak or structurally blocked for bringing GMAP-210 to the membrane. Alternatively, it is competed by endogenous GMAP-210. Depletion of endogenous GMAP-210 frees Rab1 for binding mutant GMAP-210. However, this effect is not visible in hTERT-RPE1 cells.

Figure 68: Model Of Competition Between Endogenous And GRAB Domain Mutant GMAP-210 For Rab1 Binding On The Golgi Apparatus. Endogenous GMAP-210 binds the GRAB domain binding factor “X” (allegedly Arf1), which causes a conformational change in GMAP- 210 and enables Rab1 binding. Exogenously expressed GMAP-210 is sterically hindered to bind Rab1, but can do so, if no endogenous GMAP-210 is present to compete for Rab1 on the Golgi apparatus. A factor “Y” might label a GMAP-210 specific pool of Rab1; not drawn to scale.

3.4.3 Conclusion

GMAP-210 is a large coiled-coil domain protein targeted to the Golgi apparatus by its C-terminal GRAB domain (Fig.69). Studies inS.cerevisiaeshow, that the GRAB domain of Rud3p binds to Arf1p on the Golgi apparatus. In this work, no interactor from the Arf- and Arl-family could be identified for GMAP-210 with certainty. Candidates from a yeast 2-hybrid screening with all Arf family members are not Golgi proteins but are found at the plasma membrane or do not interact with the GRAB domain. Pulldown experiments do also not confirm interaction. Arf1, however, interacts with the GMAP- 210 GRAB domain, when the minimal domain is tested in yeast 2-hybrid. This raises the possibility, that binding of Arf1 to GMAP-210 is regulated by different factors, which induce a structural change in GMAP-210 and thus expose the GRAB domain for Arf1 interaction.

210 (Fig.69). In HeLa L cells GRAB mutant GMAP-210 is recruited to the Golgi appara- tus, when endogenous GMAP-210 is depleted. It is further shown, that this recruitment is mediated by the same region Rab1 binds to. It is thus possible, that GMAP-210 interacts with a small GTPase by its GRAB domain and with Rab1 by a domain in the middle part in a synergistic way. GRAB mutants cannot compete with endogenous GMAP-210 for Rab1 binding (Fig.68). Only in the absence of endogenous GMAP-210, these GRAB mutants are recruited to the Golgi. For interaction of GMAP-210 and Arf1 a regulation of the binding domain could likewise be proposed. GMAP-210 can only interact with Arf1 when an additional factor causes a conformational change and exposes the GRAB domain for binding. This could explain, why Arf1 cannot interact with full-length GMAP- 210in vitro, but shows up in a yeast 2-hybrid experiment, which uses the minimal GRAB domain. Further testing will be necessary.