Posttranslational modifications of cause SEE COMMENTARY effective displacement of GDP dissociation inhibitor

Lena K. Oesterlina, Roger S. Goodya, and Aymelt Itzena,b,1

aDepartment of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany; and bCenter for Integrated Science Munich (CIPSM), Chemistry Department, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany

Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved January 28, 2012 (received for review December 21, 2011)

Intracellular vesicular trafficking is regulated by approximately stimulating their intrinsic GTPase activity and thereby generating 60 members of the Rab subfamily of small Ras-like GDP/GTP bind- GDP bound Rab. ing proteins. Rab proteins cycle between inactive and active states Because Rab proteins cycle not only between the GDP and as well as between cytosolic and membrane bound forms. Mem- GTP states but also between the cytosolic and membrane asso- brane extraction/delivery and cytosolic distribution of Rabs is ciated forms, another regulatory factor is necessary to compete mediated by interaction with the protein GDP dissociation inhibitor with the high binding affinity between prenylated Rabs and lipid (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. bilayers. In mammalian cells, two isoforms of the GDP dissocia- Because the Rab:GDP:GDI complex is of high affinity, the question tion inhibitor are capable of binding ca. 60 mammalian Rabs in arises of how GDI can be displaced efficiently from Rab protein in their prenylated form with high affinity, thereby extracting the order to allow the necessary recruitment of the Rab to its specific prenylated Rabs from membranes and solubilizing them in the target membrane. While there is strong evidence that DrrA, as a form of Rab:GDP:GDI complexes. The stability of these com- bacterially encoded GDP/GTP exchange factor, contributes to this plexes is only high (Kd in the nanomolar range) if the Rab protein event, we show here that posttranslational modifications of Rabs is both prenylated and in its inactive state. When active and/or can also modulate the affinity for GDI and thus cause effective dis- unprenylated, this affinity is decreased by 3–4 orders of magni- placement of GDI from Rab:GDI complexes. These activities have tude to a high micromolar Kd (2, 3). The recruitment of Rabs been found associated with the phosphocholination and adenyly- from the pool of soluble cytosolic Rab:GDP:GDI complexes to lation activities of the AnkX and DrrA/SidM, respectively, a specific membrane thus requires the dissociation of the tightly from the pathogenic bacterium Legionella pneumophila. Both bound GDI prior to the initiation of a certain vesicular transport modifications occur after spontaneous dissociation of Rab:GDI step via generation of active, GTP-bound Rabs on the membrane. complexes within their natural equilibrium. Therefore, the effec- Two models have been put forward to explain how the effective tive GDI displacement that is observed is caused by inhibition of dissociation of Rab:GDI complexes can be achieved. reformation of Rab:GDI complexes. Interestingly, in contrast to Membrane association of the two endosomal Rab proteins adenylylation by DrrA, AnkX can covalently modify inactive Rabs Rab5 and Rab9 is accompanied by exchange that oc- with high catalytic efficiency even when GDP is bound to the curs shortly after membrane association (4, 5). These results led GTPase and hence can inhibit binding of GDI to Rab:GDP com- to the assumption that a GDI displacement factor (GDF) could plexes. We therefore speculate that human cells could employ si- influence the release of prenylated Rab proteins from GDI (6). milar mechanisms in the absence of infection to effectively displace The only identified GDI displacement factor to date is Yip3/ Rabs from GDI. PRA1 (7, 8). Yip3/PraI can inhibit the extraction of Rab3 from membranes (7) and furthermore has been shown to influence the Legionnaires’ disease ∣ Rab recruitment ∣ RabGDI dissociation of a Rab9:GDI complex and to facilitate the mem- brane recruitment of Rab9 from its complex with GDI in in vitro he intracellular activity of most signaling proteins is regulated extraction assays (8). In vivo, Yip3/PRA1 depletion by RNAi Tat the temporal and spatial level. In the regulation of vesicular leads to reduced membrane association of Rab9 (8). Based on trafficking, small Ras-like of the Rab subfamily are con- these data a GDF would be defined as a protein that can influ- trolled in their distribution between the cytosol and intracellular ence the equilibrium between GDI bound and free Rab proteins. membranes, which is in turn coupled to the binding to either Recently, we have demonstrated that the GEF domain of GDP or GTP. Cycling between the cytosol and membranes is de- DrrA is sufficient to effectively displace GDI from Rab:GDP: pendent on the activation state of the Rab protein. In the active GDI complexes by generating the GTP state of Rab proteins after GTP-bound form, Rabs are associated with membranes, where complex dissociation, which has only low affinity for GDI (3, 9). they interact with effector proteins that promote diverse steps It has been shown that the protein DrrA/SidM secreted by the in vesicular trafficking. When bound to GDP, Rabs are defined intracellular bacterial pathogen Legionella pneumophila can effi- as inactive and are predominantly distributed in the cytosol ciently recruit Rab1 to the Legionella containing vacuoles in bound to GDI. In order to associate with the cytosolic surface which the bacterium resides and replicates intracellularly (10, 11). of intracellular membranes, most Rab proteins possess two hy- Rab1 recruitment and GDI displacement is exerted by the GEF- drophobic prenyl (geranylgeranyl) moieties at their structurally activity of DrrA (9), suggesting that mammalian GEFs may also flexible C terminus that are posttranslationally attached via the be capable of activating and thereby recruiting Rab proteins from concerted action of Rab geranylgeranyl transferase (RabGG- Tase) and Rab escort protein utilizing geranylgeranyl pyropho- Author contributions: R.S.G. and A.I. designed research; L.K.O. conducted experiments; sphate as a cosubstrate. These geranylgeranyl groups mediate L.K.O., R.S.G., and A.I. analyzed the data; and L.K.O., R.S.G., and A.I. wrote the paper. stable peripheral binding of Rabs to a cognate membrane by in- The authors declare no conflict of interest. serting the hydrophobic anchors into the lipid bilayer (1). Activa- This article is a PNAS Direct Submission. tion of Rab proteins is catalyzed by GDP/GTP exchange factors See Commentary on page 5555. (GEFs) that displace the tightly bound GDP by the excess of 1To whom correspondence should be addressed. E-mail: aymelt.itzen@mpi-dortmund. GTP from the cellular environment. Because most active Rab mpg.de. proteins do not hydrolyze GTP on a physiologically relevant time This article contains supporting information online at www.pnas.org/lookup/suppl/ BIOCHEMISTRY scale, GTPase activating proteins assist in deactivating Rabs by doi:10.1073/pnas.1121161109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1121161109 PNAS ∣ April 10, 2012 ∣ vol. 109 ∣ no. 15 ∣ 5621–5626 Downloaded by guest on September 25, 2021 their complexes with GDI to membranes where specific GEFs are the influence of these modifications on the association with GDI. localized. Stopped-flow analysis revealed that the presence of the AMP- This model requires that the Rab protein dissociates from GDI group on Tyr77 of Rab1b completely blocked binding of Rab1b- before activation by GEFs. However, the intrinsic rate of GDI AMP-f-NBD to GDI (Fig. 2B). A slight increase in fluorescence dissociation is low due to the high affinity between prenylated was indicative of a small amount of nonadenylylated Rab1b-f- Rab:GDP and GDI (3, 9, 12) and hence limits the rate at which NBD as a contaminant, as demonstrated by mass spectrometry active Rab can be generated. Thus, GDI dissociation can poten- (Fig. 2A). Furthermore, determining the association rate constant tially become rate limiting for the initiation of vesicular traffick- of the minor phase showed that this contaminant behaved exactly ing processes. In addition to the possibility that bona fide GDF like nonadenylylated Rab1b-f-NBD (Fig. 2C). Thus, the presence molecules exist that can accelerate this process, we wondered of the AMP group on Rab1b-AMP-f-NBD blocked GDI-binding whether inactive Rab bound to membranes could be generated entirely. Because no interaction with GDI can be detected under by different mechanisms and that this pool of Rab:GDP could these experimental conditions, the Kd between GDI and Rab1b- be mobilized rapidly. Here we show that two posttranslational AMP-f-NBD must be higher than 225 μM (see SI Text for addi- modifications (by AnkX and DrrA) can drastically reduce the tional explanations). Thus, adenylylation of Rab1b lowers the affinity of different Rab proteins for GDI. Furthermore we show affinity for GDI by at least five orders of magnitude. that AnkX can dissociate Rab:GDI complexes efficiently, be- Binding of GDI to phosphocholinated Rab1b-f-NBD was cause it modifies even inactive, GDP-bound Rab proteins that also tested using the described fluorescent assay. As seen for the spontaneously dissociate from the Rab:GDP:GDI complex with adenylylated form of Rab1b-f-NBD, no interaction with GDI high catalytic activity and inhibits their rebinding to GDI via this was seen under the chosen experimental conditions (Fig. 2D). modification. Hence, phosphocholination on Ser76, like adenylylation on Results Tyr77, appears to decrease the affinity for GDI by several orders It has been described recently that enzymes from Legionella of magnitude. covalently modify human Rab1 and Rab35. Legionella AnkX transfers a moiety from CDP-choline specifically Effective GDI Displacement by Binding Competition. We have demon- on Ser76 of Rab1b or Thr76 of Rab35 by enzymatic action of its strated previously that nucleotide exchange from GDP to GTP FIC domain (Fig. 1A) (13). DrrA contains an N-terminal adeny- is a way of effectively displacing GDI from Rab proteins (3, 9). lyltransferase domain (Fig. 1A) capable of covalently attaching AMP to the hydroxyl function of Rab1b Tyr77 (14). Interestingly, A 1.6 B the modified residues are located centrally in the complex inter- Rab1b-AMP face with GDI (Fig. 1B) suggesting that the bulky phosphocholine 1.4

(PC) and AMP groups might interfere with GDI binding. 1.2 Rab1b-f-NBD Rab1b Rab1b-AMP-f-NBD

Inhibition of GDI Binding by Posttranslational Modifications. Rab1b norm. fluorescence 1 and Rab35 were prenylated with the fluorescent geranylgeranyl 22,600 22,800 23,000 23,200 00.02 0.04 0.06 mass / Da time / s analogue farnesyl-NBD (7-nitrobenz-2-oxa-1,3-diazole) (f-NBD) 200 C that can report on binding of Rab proteins to GDI by a substan- 150 -1

tial increase in NBD fluorescence (3). Subsequently, by employ- / s 100 obs ing the enzymatic activity of AnkX and DrrA, Rab1b-f-NBD were k 50 Rab1b-AMP-f-NBD phosphocholinated or adenylylated, respectively, to investigate Rab1b-f-NBD 0 1,000 1,200 1,400 1,600 1,800 2,000 02010 30 A AnkX m / z [GDI-1] / µM FIC A1A2 A3 A4 1 155 289 424 620 690 994 D 3.0 391 460 588 658 1: 50 nM Rab1b-f-NBD 2.5 25 nM AnkX DrrA (1 mM CDP-Choline) * * 2.0 2: + 75 nM GDI-1 Adenylyltransferase GEF P4M 1.5 1 340 533 647 1 2 1.0 B + CDP-Choline 0.5 - CDP-Choline normalized fluorescence 0.0 40200 60 80100 120 140 200180160 time / s Ser-PC Fig. 2. GDI binding to adenylylated and phosphocholinated Rab1. (A) Electrospray ionization mass spectrometry (ESI-MS) analysis of Rab1b after preparative adenylylation with DrrA. Approximately 4% of Rab1b is not adenylylated. (B) Binding of 25 μM GDI-1 to 0.5 μM Rab1b-AMP-f-NBD and Rab1b-f-NBD monitored by measurement of NBD fluorescence. The am- Tyr-AMP plitude of the fluorescence change is reduced in adenylylated Rab but traces show identical time dependence. Therefore both reactions can be associated with binding of unmodified Rab1b-f-NBD to GDI. (C) Association of adeny- Fig. 1. Phosphocholination and adenylylation of Rab1. (A) Domain architec- lylated and nonadenylylated Rab1b-f-NBD (50 nM) with varying concentra- ture of AnkX (30) and DrrA (9, 14). AnkX contains an N-terminal FIC domain tions of GDI-1 was measured via stopped-flow by monitoring the change in and four ankyrin repeat regions (A1-A4). DrrA is comprised of an adenylyl- NBD-fluorescence. Rate constants were determined by single exponential fit transferase domain, a GEF domain and a phosphatidylinositol-4-phosphate of the observed fluorescence change and plotted against GDI-I concentra- binding domain (P4M). (B) Surface representation of a model of adenylylated tion. (D) Binding of GDI (75 nM) to phosphocholinated and nonphosphocho- Rab1b (14) after phosphocholination. The geometry of the phosphocholine linated Rab1b-f-NBD (50 nM). Phosphocholination with AnkX (1 μM) was group is chosen arbitrarily. The surface areas of Rab1 that likely interact with carried out in situ in the fluorescence cuvette. No GDI-1 binding to Rab1b- GDI are colored in green [based on the structure of the Rab1 homologue Ypt1 PC-f-NBD can be observed. As a negative control Rab1b-f-NBD was incubated from yeast in complex with yeast GDI (31)]. in the absence of CDP-choline.

5622 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1121161109 Oesterlin et al. Downloaded by guest on September 25, 2021 Motivated by recent findings, we investigated the disruption subsequently adenylylated, thereby shifting the equilibrium to

of GDI:Rab:GDP complexes by proteins with high affinity to quantitative GDI displacement. SEE COMMENTARY GDP-bound Rabs. The Legionella protein LidA has a Kd for To show that nucleotide exchange is important for adenylyla- Rab1 in the subnanomolar range (15) and based on a comparison tion of Rab1b, but that it is not important to have the GEF activity of the binding surfaces of LidA and GDI on Rabs should there- on the same protein, the same reaction was performed with a fore efficiently compete with GDI-binding to Rab1b, which is of GEF-deficient DrrA quadruple mutant [N451A/R453A/D480S/ nanomolar affinity (3). We confirmed this hypothesis by displa- S483A, (9)] (Fig. 3B), demonstrating no GDI displacement. GDI cing GDI from the complex with Rab1b-f-NBD (50 nM) by ad- was effectively displaced only after addition of the DrrA GEF dition of LidA (1 μM) (Fig. 5B). Thus, GDI can be displaced by domain (DrrA340–533), demonstrating that Rab1 activation by GDP/GTP-exchange, but also by competition with Rab binding nucleotide exchange is needed to facilitate DrrA-catalyzed proteins of high affinity. The determined rate constant for adenylylation. GDI displacement by LidA corresponds to the intrinsic dissocia- We have shown recently that the ATase activity of DrrA is tion constant of Rab1b-f-NBD [0.0279 s−1 (3)] showing that specific for a subset of Rab proteins (14): Rab8a and its yeast LidA is not capable of increasing the dissociation rate constant homologue Sec4 are adenylylated by DrrA in vitro whereas e.g., of the Rab:GDI complex, but simply binds to dissociated Rab Rab5 is not. The combination of activation of Rab1b-f-NBD by protein after dissociation from GDI. nucleotide exchange with adenylylation by DrrA effectively dis- places GDI (Fig. 4A). Rab5a-f-NBD is not a substrate for DrrA Effective GDI Displacement by Adenylylation. We have seen that and consequently forms a DrrA ATase resistant GDI-complex, GDI can be effectively displaced from its complex with Rabs even in the presence of Rab5-GEF domain [Rabex5132–397, (16)] either by GDP/GTP exchange or by direct competition (LidA), (Fig. 4B). However, GDI displacement by adenylylation of because in both cases reformation of the complex is inhibited. Sec4-f-NBD combined with the GEF-activity of the Sec4-GEF The loss of GDI binding to Rab1 due to adenylylation suggested Sec2 [Sec21–165, (17)] shows a very similar behavior compared that posttranslational modification might be a further mechanism to Rab1b-f-NBD (Fig. 4C). When repeating the experiment with to effectively release Rab proteins from GDI on the basis of an the GEF-domain of the human Sec2-homologue Rabin8 enzymatic reaction by precluding Rab:GDI complex reformation (Rabin8153–237), the adenylylation reaction and thus the displace- after dissociation. To investigate the dynamics of this effect, the ment of GDI becomes rate limited by Sec4-f-NBD activation Rab1b-f-NBD:GDI complex was generated in a fluorescence cuv- (Fig. 4D), probably because Rabin8 is a weaker GEF for Sec4 ette (Fig. 3A). The addition of DrrA and ATP in the presence of excess GDP was unable to catalyze GDI displacement. However, 1.2 1: 50 nM Rab1b-f-NBD when GTP was added at a concentration 10x less than GDP,GDI- A 123 75 nM GDI-1 1.0 1 µM DrrA340-533 dissociation is readily seen. 0.8 100 µM GTP, 1 mM GDP 2: + 1 µM DrrA The complete displacement of GDI under these experimental fl 0.6 3: + 100 µM ATP conditions is mediated by adenylylation of active Rab1b-f-NBD 0.4 but not by GTP-loading itself, because only approximately 10% of 0.2

Rab1b-f-NBD (GTP:GDP ratio of 1∶10) is activated. However, normalized fluorescence 0 0 500250 750 1,000 1,250 1,500 1,750 2,000 because active Rab1b is a 300-fold better substrate for the ATase time / s domain of DrrA than the inactive form (14), the presence of 1.2 B 12 3 small concentrations of free GTP in the experiment constantly 1.0 1: 25 nM Rab5a-f-NBD generates active Rab1b via the GEF activity of DrrA that is 0.8 37.5 nM GDI-1

0.6 1 µM Rabex5132-397 100 µM GTP, 1 mM GDP 0.4 2: + 1 µM DrrAfl N451A/R453A/ A 1.2 1234 5 0.2 D480A/S483A 1:+ 50 nM Rab1b-f-NBD, 75 nM GDI-1 3: + 100 µM ATP 1.0 2: + 1 mM GDP normalized fluorescence 0 3: + 1 µM DrrA 0250 500 750 1,000 1,250 1,500 fl time / s 0.8 4: + 100 µM ATP 5: + 100 µM GTP 1.1 0.6 C 12 1: 25 nM Sec4-f-NBD 1.0 37.5 nM GDI-1 1 µM His-Sec2 0.4 0.9 1-165 3 100 µM GTP, 1 mM GDP 0.8 0.2 2: + 1 µM DrrAfl N451A/R453A/ 0.7 D480A/S483A normalized fluorescence 0 3: + 100 µM ATP 0500 1,000 1,500 2,000 2,500 0.6

time / s normalized fluorescence 0.5 0500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 B time / s 1.2 1234 5 6 1: + 50 nM Rab1b-f-NBD, 75 nM GDI-1 1.0 2: + 1 mM GDP 1.1 1: 25 nM Sec4-f-NBD, 37.5 nM GDI-1, 3: + 1 µM DrrA N451A/R453A/D480A/S483A D fl 12 1 µM Rabin8153-237, 100 µM GTP, 1 mM GDP 4: + 100 µM ATP 2: + 1 µM DrrA N451A/R453A/D480A/S483A 0.8 1.0 fl 3: + 100 µM ATP 4: + 1 µM His-Sec2 5: + 100 µM GTP 3 1-165 6: + 1 µM DrrA 0.6 340-533 0.9 4 0.4 0.8 0.2

normalized fluroescence 0.7 normalized fluorescence 0 0 2,000 4,000 6,000 8,000 10,000 12,000 0500 1,000 1,500 2,000 2,500 3,000 time / s time / s Fig. 4. GDI displacement from different Rab:GDI complexes by adenylyla- A C D Fig. 3. GDI displacement from Rab1b by adenylylation. GDI displacement by tion. Adenylylation of Rab1b-f-NBD ( ) and Sec4-f-NBD ( , ) by DrrAfl A adenylylation with DrrAfl ( ) or GEF-mutant DrrAfl N451A/R453A/D480A/ N451A/R453A/D480A/S483A. In the absence of ATP the Rabs are modified S483A (B). Complex dissociation is monitored by the change in NBD fluores- with GMP by the ATase domain of DrrA (14). Rab5a (B), which is not a sub- cence measurement. In this assay, release of GDI-1 from Rab1b is dependent strate of the ATase domain of DrrA cannot be displaced by DrrA GEF mutant. on nucleotide exchange to GTP. In the presence of DrrAfl (A), GDI is released The rate of GDI displacement is lower in the presence of Rabin8153–237 com- BIOCHEMISTRY after addition of GTP whereas in the presence of DrrA GEF mutant (B) addi- pared to Sec21–165, which corresponds to the lower nucleotide exchange tion of active DrrA GEF domain (DrrA340–533) is needed for GDI release. activity of Rabin8153–237 for Sec4.

Oesterlin et al. PNAS ∣ April 10, 2012 ∣ vol. 109 ∣ no. 15 ∣ 5623 Downloaded by guest on September 25, 2021 1.2 1234 1: 50 nM Rab35-f-NBD than Sec2. The addition of additional Sec2 then accelerates ade- A 2: + 75 nM GDI-1 1.0 3: + 1 mM CDP-Choline nylylation and thus GDI displacement (Fig. 4D, addition #4). 4: + 250 nM AnkX Thus, the effective release of GDI from a complex with Rab 0.8 proteins caused by adenylylation of dissociated Rab proteins is 0.6 possible but needs to be facilitated by previous Rab activation, 0.4 because active Rabs are 300-fold better substrates for DrrA com- 0.2 pared to inactive forms (14). normalized fluorescence 0.0 03,000500 1,000 1,500 2,000 2,500 3,500 4,000 4,500 time / s 1 2 Effective GDI Displacement by Phosphocholination. The finding that 1.1 121: 25 nM Rab35-f-NBD, C 1.1 1: 50 nM Rab35-f-NBD, B 1.0 37.5 nM GDI-1 1.0 adenylylation can promote GDI displacement from Rab proteins 2: + 1 µM LidA 75 nM GDI-1 0.9 0.9 1 mM CDP-Choline motivated us to perform similar experiments on the effect of 2: + 1 µM AnkX phosphocholination by the Legionella protein AnkX (13). We uti- 0.8 0.8 lized the fluorescence based assay described above to monitor 0.7 0.7 0.6 0.6 the binding and dissociation kinetics of Rab1b-f-NBD to GDI. k = 0.0020 s-1 k = 0.0022 s-1 0.5 off 0.5 obs normalized fluorescence

The addition of CDP-choline and AnkX to a Rab1b-f-NBD: normalized fluorescence 0.4 0.4 GDP:GDI complex caused GDI dissociation (Fig. 5A). This 0 500 1,000 1,500 2,000 0 500 1,000 1,500 2,000 effect could be reversed by adding the lpg0696 which time / s time / s was shown to reverse the phosphocholination reaction by hydro- Fig. 6. GDI displacement by phosphocholination of Rab35. (A) GDI can be lytically removing the phosphocholine group from Rab1b (18), effectively displaced from Rab35-f-NBD by phosphocholination with AnkX. thereby reconstituting the Rab1b-f-NBD:GDP:GDI complex. (B) Single exponential fit of GDI displacement by LidA showing the intrinsic The addition of the tightly binding LidA was again able to dis- dissociation rate of the Rab35-f-NBD:GDI complex. (C) GDI displacement by place GDI. phosphocholination with AnkX. The observed rate constant is lower than the intrinsic dissociation rate of the complex. Adenylylation or GTP-loading of Rab1b-f-NBD by DrrA can- not accelerate GDI displacement beyond the intrinsic rate of GDI dissociation. Thus, we investigated whether phosphocholi- on a physiologically meaningful time scale, indicating that cova- nation could promote GDI displacement faster than the sponta- lent modification of Rab proteins is a way to effectively dissoci- neous rate of GDI dissociation. Tothis end we compared the GDI ate GDI. displacement by LidA binding (Fig. 5B) with the displacement caused by the enzymatic activity of AnkX (Fig. 5C). The displa- Discussion k ¼ 0 0277 −1 cement by LidA ( off . s ) was twice as fast as the dis- The binding of Rab proteins to the universal regulatory protein sociation caused by Rab1b-f-NBD phosphocholination with GDI is of crucial importance for Rab membrane cycling and thus k ¼ 0 0150 −1 AnkX ( obs . s ) under the conditions used. for the regulation of vesicular trafficking (19). In order to exert Because Rab35 is also a substrate for AnkX (13), we per- their function, Rab proteins need to be recruited specifically to formed similar experiments with Rab35-f-NBD (Fig. 6 A–C). the cytosolic surface of intracellular membranes in a temporally GDI displacement from Rab35-f-NBD:GDP by phosphocho- controlled manner. However, the affinity of the interaction be- k ¼ 0 0022 −1 k ¼ lination ( obs . s ) was as fast as with LidA ( off tween prenylated Rabs and GDI is high and consequently the 0.0020 s−1). Therefore, GDI displacement of Rab35-f-NBD: spontaneous complex dissociation is very slow. Therefore, me- GDI by phosphocholination is limited by the intrinsic rate of chanisms for regulated recruitment of Rabs and hence effective GDI dissociation. displacement of GDI from cytosolic Rab:GDP:GDI complexes Hence, in contrast to adenylylation by DrrA, GDI displace- must exist. In earlier works, it was shown that human Pra1 and ment from inactive Rab1b-f-NBD:GDP by AnkX was possible its yeast homolog Yip3 act as GDI displacement factors and are capable of recruiting prenylated Rab9:GDP to membranes prior

1.2 1: 50 nM Rab1b-f-NBD to activation by nucleotide exchange (Fig. 7A). However, the A 1 2 43 6 2: + 75 nM GDI-1 mechanism of the GDI displacement reaction remains elusive. 1.0 3: + 1 mM CDP-Choline 4: + 250 nM AnkX Recently, it was shown that nucleotide exchange itself makes 0.8 5: + 500 nM lpg0696 6: + 1 µM LidA an important energetic contribution to the effective displacement 0.6 of GDI (3, 9), and thus that GEFs could potentially recruit Rab 5 0.4 proteins to membranes without the need for an additional GDI 0.2

normalized fluorescence displacement factor (Fig. 7B). Nevertheless, this mechanism of 0.0 02,000500 1,000 1,500 2,500 3,000 3,500 4,000 GDI displacement is incapable of accelerating the slow GDI dis- time / s sociation, which means that the rate at which Rab proteins could 1.2 1.2 BC12 21 be recruited to membranes would be limited by the spontaneous 1.0 1.0 1: 50 nM Rab1b-f-NBD, 1: 50 nM Rab1b-f-NBD, rate of dissociation of GDI:Rab:GDP complexes in the absence 0.8 75 nM GDI-1 0.8 75 nM GDI-1 2: + 1 µM LidA 1 mM CDP-Choline of additional factors. 0.6 0.6 2: + 2 µM AnkX In this work, we investigated the influence of posttranslational 0.4 0.4 modifications of Rab proteins on the ability to displace GDI from

0.2 -1 0.2 -1 prenylated Rabs. It has been observed that the enzymes DrrA and koff = 0.0277 s kobs = 0.0150 s normalized fluorescence 0.0 normalized fluorescence 0.0 AnkX from Legionella can adenylylate and phosphocholinate 0 100 200 300 400 0 200 400 600 800 time / s time / s Rab1b and Rab35 at Tyr77 and Ser76, respectively. As demon- strated here, these modifications abolish GDI binding and hence Fig. 5. GDI displacement by phosphocholination of Rab1b. (A) GDI can be lead to effective displacement of GDI from the Rab protein. GDI effectively displaced from Rab1b-f-NBD by phosphocholination with AnkX. displacement by adenylylation, however, was dependent on the Reversal of phosphocholination by lpg0696 leads to an increase in fluores- cence that corresponds to rebinding of GDI as addition of LidA needs to a activation of Rab1 by nucleotide exchange because the catalytic rebinding of GDI. (B) Single exponential fit of GDI displacement by LidA activity of DrrA for Rab1b:GDP is low (14). In contrast, the cat- showing the intrinsic dissociation rate of the Rab1b-f-NBD:GDI complex. alytic activity of AnkX is high for both GDP- and GTP-bound (C) GDI displacement by phosphocholination with AnkX. The observed rate Rab1 (and Rab35) and thus the enzymatic activity of AnkX is constant is lower than the intrinsic dissociation rate of the complex. sufficient to displace GDI. As found for GDI displacement by

5624 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1121161109 Oesterlin et al. Downloaded by guest on September 25, 2021 SEE COMMENTARY

Fig. 7. Conceivable GDI displacement mechanisms. Forced or spontaneous GDI-dissociation are prerequisites for membrane association of Rab proteins. In the classical mechanism (A), PraI recruits Rab proteins to the membrane; but it is unclear whether PraI interacts with GDI-free or GDI-bound Rab. Another recruitment mechanism is based on RabGEFs that displace GDI by exploiting its low affinity for active, GTP-bound Rabs (B). Whether this process operates on cytosolic or membrane-bound Rabs is unclear. Here it has been found that the displacement of GDI from Rab proteins by posttranslational modificationsis another possibility for Rab membrane recruiting (C). Similar to GEFs, phosphocholination by AnkX could happen on cytosolic or membrane-bound Rabs. By this mechanism, a membrane-bound pool of inactive Rabs can be generated that can be utilized by another enzyme (here lpg0696) for downstream processes such as activation by GEFs. In this manner, a slow Rab GDI-dissociation step can be replaced by a faster enzymatic process; i.e., dephosphocholination, when fast mobilization and activation of a Rab protein is needed.

nucleotide exchange (3, 9), the rate of complex disruption caused existence of eukaryotic enzymes that modify Rab proteins in or- by phosphocholination is limited by the intrinsic dissociation der to suppress GDI binding and to localize Rabs to intracellular rate of Rab:GDI complexes. Hence, neither phosphocholination membranes prior to activation by GEFs is conceivable but none nor nucleotide exchange actively displace GDI from Rab because have been reported thus far. they do not accelerate the rates of dissociation of the complexes. We have shown that adenylylation and phosphocholination of Nevertheless, disruption of the Rab:GDP:GDI complex forma- Rab proteins are able to modulate the affinity for the regulator tion by covalent modifications might solve the thermodynamic protein GDI. The enzyme AnkX can cause effective GDI displa- problem of slow complex dissociation even if they cannot accel- cement from Rab1 and Rab35 by phosphocholinating these Rabs. erate the process. For example, phosphocholination of Rab1 by GDI displacement is limited by the rate of spontaneous Rab GDI AnkX will disrupt GDI binding and thus lead to incorporation of complex dissociation only, therefore it is similar to the GDI dis- Rab1 into membranes where AnkX resides (Fig. 7C). In this placement by GEFs in vitro (3, 9). Consequently, posttransla- state, Rab1 is membrane bound in the inactive state but is resis- tional modifications of Rab proteins could represent a way to tant to activation by GEFs (13). This scenario would lead to a recruit Rabs to a particular membrane prior to their activation stable membrane pool of GDP-bound Rab, with serious conse- by nucleotide exchange. quences for transport mechanisms. The action of a demodifying enzyme, lpg0696 in this case, renders Rab1 accessible to GEFs Materials and Methods again so that vesicular trafficking could proceed. The rate of gen- Protein Isolation. Sec4, Rab5a, Rab35, and codon-optimized Rab1b (Gene, eration of active Rabs would then become dependent on the de- Regensburg, Germany) were C-terminally modified to contain the GGTase modification reaction instead of the dissociation of Rab:GDI I recognition sequence CVIL using the QuikChange mutagenesis kit (Stratagene). Protein expression was performed in BL21(DE3) (Rab1b, complexes. Therefore, posttranslational modifications of Rab Rab5a, DrrA) or BL21-CodonPlus(DE3)-RIL cells (Rab35, Sec4, AnkX, lpg0696, proteins could potentially generate a stable pool of GDP-bound GGTase I) at 20 °C over night or at 37 °C for 5 h (Rab5a). Protein expression Rabs that can be quickly utilized for subsequent steps. Posttran- was induced with 0.2 mM, 0.3 mM (Sec4, LidA) or 0.5 mM IPTG (GGTase I). slational modifications might represent a general mechanism of Rab1b, Rab5a, Rab35 were expressed as cleavable His6-MBP-fusion con- controlling GDI binding and the cytosolic or membrane distribu- structs, Sec4 and Rabin8 were expressed as cleavable His6-fusion constructs. tion of Rab proteins. In agreement with this argument, it has been GGTase I α and β were coexpressed. GGTase I α was expressed as a His6-GST reported that several reactions can modify the fusion construct and GGTase I β as a His6-fusion construct. Bovine GDI-I membrane-cytoplasm partitioning of Rab proteins. Rab1a and was expressed in Sf21 insect cells using the Bac-to-Bac Expression System Rab4 are modified during mitosis by Cdk1 (20). The cytoplasmic (Invitrogen) with a cleavable His-tag. Protein purification was performed analogous to the isolation procedure concentration of Rab4 is increased upon phosphorylation (20, 21) described for Rab8 (but without ATP) (26). Isolation of Rabin8, Rab5a, and whereas phosphorylation of Rab1 leads to a decrease in cytoplas- Rab35 was performed at pH 7.5 and Rabin8, DrrA, AnkX, and lpg0696 were mic protein (20). Phosphorylation of Rab6 by Protein Kinase C isolated without GDP and MgCl2. To stabilize the proteins for Rab5a, AnkX, also decreases the cytoplasmic protein concentration (22). and lpg0696 the NaCl concentration was increased to 200 mM. Additionally Furthermore, it could be shown that Rab4 and Rab5 associated Rab5a was isolated in the presence of 5% glycerol. Residual MBP-tag con- GDI molecules are phosphorylated in vivo (23), but it remains taminants were removed via an amylose resin. GGTase I was isolated as a fu- unclear which residues are phosphorylated. phosphoryla- sion protein; therefore the protease cleavage and the second Ni-IDA chromatography steps were omitted. The first Ni-IDA chromatography with tion of GDI-1 by serum- and glucocorticoid-inducible kinase 1 β (24) as well as phosphorylation on GDI-2 increases the GDI-I was performed with 50 mM NaPi pH 8, 300 mM NaCl and 2 mM -Mer- captoethanol and 5 mM β-Mercaptoethanol was used instead of DTE amount of cytoplasmic Rab4:GDI complex (25). These differ- (dithioerythritol) during the other purification steps. His6-Sec21–165 and LidA ences in membrane-cytoplasm partitioning could be caused by were expressed and isolated as described (17, 27). Rabex5132–397 was cloned BIOCHEMISTRY a modification in Rab:GDI affinity similar to the mechanisms by the Dortmund Protein Facility into a pOPINF-vector (N-terminal His6-tag of phosphocholination and adenylylation described here. The followed by a PreScission protease cleavage sequence) by the in-fusion

Oesterlin et al. PNAS ∣ April 10, 2012 ∣ vol. 109 ∣ no. 15 ∣ 5625 Downloaded by guest on September 25, 2021 cloning method (28). Rabex5132–397 was expressed in Escherichia coli BL21 Kinetic Measurements. Fluorescence measurements were performed with a (DE3) RIL 4 h at 37 °C after induction with 1 mM IPTG and purified by a com- FluoroMax-3 (Horiba Jobin Yvon Inc.) or a stopped-flow apparatus (Applied bination of Ni-IDA and size exclusion chromatography in a final buffer con- Photophysics). NBD fluorescence was excited at 479 nm and emission was de- sisting of 25 mM Hepes, pH 7.5, 40 mM NaCl and 1 mM TCEP. tected at 525 nm. For stopped-flow measurements fluorescence was excited at 437 nm and emission was detected through a 530 nm cut off filter. Mea- . NBD-farnesyl pyrophosphate was produced as described else- surements were performed in 25 mM Hepes pH 8, 50 mM NaCl, 5 mM MgCl2 where (29). Prenyltransferase, Rab protein and substrate were mixed in a and 5 mM DTE at 25 °C. 0.5∶1:5ratio and incubated at room temperature for 1.5 h. GGTase I and farnesyl-NBD were preincubated for 30 min before addition of Rab protein. Preparative Adenylylation. Rab1b, DrrAfl, and ATP were mixed in a 10∶1∶25 Prenylated Rab1b was separated from GGTase I by several rounds of GSH and ratio and incubated for 1.5 h at room temperature. DrrAfl was removed by Ni-IDA affinity chromatography in the presence of 5% CHAPS with a final buffer containing 20 mM Hepes pH 7.2, 50 mM NaCl, 2 mM β-Mercaptoetha- gel filtration chromatography using a Superdex 75 column equilibrated in μ nol, 2 mM MgCl2 and 10 μM GDP. Rab5a, Rab35, and Sec4 were separated 20 mM Hepes pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE and 10 M GDP. from GGTase I with Gel filtration chromatography using a Superdex 200 10∕30 column with 20 mM Hepes pH 7.2, 500 mM NaCl, 2 mM MgCl2, ACKNOWLEDGMENTS. Nathalie Bleimling is acknowledged for invaluable 2 mM DTE and 10 μM GDP. The NaCl concentration in the reaction mixture technical assistance. L.K.O. was supported by the International Max Planck was increased to 500 mM and 5% CHAPS (wt∕vol) was added to the reaction Research School in Chemical Biology (Dortmund). This work was supported before loading. by a grant from the Deutsche Froschungsgemeinschaft SFB642, project A4.

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