RAF Kinases and Endocytosis Figure 2. Physiological effects of AR149 overexpression in yeast. A. Cells expressing high amounts of GFP-AR149 fail to form cortical actin patches. Cells expressing GFP-A-RAF or GFP-AR149 were grown, fixed as described in ‘‘Experimental procedures’’. Polymerized actin was visualized with Alexa Fluor 546-conjugated Phalloidin. Note that cells expressing higher levels of GFP-AR149 but not of full length A-RAF (bright green cells marked with arrows) lost polymerized actin. B. GFP-AR149 expression inhibits /-factor induced shmoo formation. Cells transformed with pUG36- AR149 (right) or A-RAF(303–606) (left) were treated with /-factor for 2 h. Note increased overall size and absence of shmoos in cells expressing GFP- AR149. C. Changes in nuclear morphology of GFP-AR149 expressing cells. DNA of the nucleus and mitochondria was stained with DAPI. Morphology of nuclei of AR149 expressors varied from fragmented (left) to completely dispersed (right). D. FM-4-64 uptake is affected in AR149-expressing yeast. Yeast transformed with pUG36-AR149 and untransformed control were incubated with lipophilic styryl dye FM 4–64 for indicated time, washed and observed by fluorescence microscopy. Transition of the red fluorescence from periplasmic endocytic sites to vacuoles is clearly visible at 20 minutes in control cells, but not in GFP-AR149 expressing cells. E. Growth inhibition of AR149 expressing cells. Cells transformed with pUG36, pUG36-A-RAF or pUG36-AR149 were grown under selective conditions in fresh selective medium. Cell proliferation was monitored by spectrophotometry. doi:10.1371/journal.pone.0004647.g002 The cells were pre-incubated in serum-free medium for 1 h at 37uC. For continuous Tfn uptake, the cells were incubated in internalization medium (HBSS medium plus 1% BSA) containing 5 mg/ml Alexa Fluor 546-conjugated human Tfn (Invitrogen) at 37uC for indicated time. After Tfn internalization the cells were extensively washed three times with ice-cold PBS and fixed with 3.7% PFA. Yeast strains and techniques Protease deficient strains cI3 ABYS 86, BJ 5459 (Yeast Genetic Stock Center, University of California, Berkeley) were used in order to prevent degradation of expressed proteins. Standard protocols for yeast growth, transformation and manipulations were employed [21]. Yeast transformation was performed by a modified litihum acetate method. The following plasmids were used in this study: pUG36 and pUG36 for detection of GFP-fusion proteins; pEG-KT for galactose-inducible expres- sion of GST-fusion proteins. Membrane fractionation on 20–35% sucrose gradients and indirect immunofluorescence microscopy were done as described previously [22]. Immunoprecipitation of GFP-AR149 and ARF6 COS7 cells were transfected with GFP-AR149 and HA- ARF6wt, HA-ARF6(Q67L) or HA-ARF6(T27N) respectively with jetPEI (Biomol). After 24 h, cells were lysed in ARF6 lysis buffer (50 mM Tris-HCl pH 7.0, 2 mM MgCl2, 100 mM NaCl, 10% Glycerol, 0.75% NP-40) containing protease inhibitors. To avoid high signal from heavy and light chains of antibodies on the Western blot we used Mouse IgG TruhBlot Set (NatuTec) including beads and secondary HRP-conjugated antibody. The clarified lysates were divided into two equilibrated parts, each of them was incubated for 2 h at 4uC with anti-GFP or anti-HA antibody respectively (Santa Cruz Biotechnology, USA). Next, the probes were precipitated with mouse-TruhBlot agarose (NatuTec) for 1 h at 4uC. Beads were washed three times in the lysis buffer with 0.2% NP-40 and protease inhibitors. GGA3 pulldown assay Activated ARF6NGTP was monitored by binding to its effector GGA3 as described previously [23]. Briefly, COS7 cells were co- transfected with HA-ARF6 plus indicated plasmids using jetPEI and grown for 24 h. EGF stimulation was performed after 24 h starvation in 0.03% serum with 100 ng/ml EGF (Cell System Biotechnologie Vertrieb) for 10 min at 37uC. Clarified lysates were incubated with 25 mg of GST-GGA3 immobilized on glutathione- Sepharose beads for 2 h at 4uC. The beads were washed three times with PBS, resuspended in SDS PAGE loading buffer and boiled. Bound proteins were size-fractionated by SDS-PAGE and detected by immunoblotting. Results Isotype specific distribution of RAF proteins in yeast All three human RAF genes were fused with the C-terminus of green fluorescent protein (GFP) and expressed under the control of a moderately inducible MET25 promoter in S. cerevisiae. GFP-B- RAF and GFP-C-RAF fluorescence was evenly distributed in the cytosol of induced cells. In contrast, A-RAF fluorescence localized to distinct punctate cortical structures, which were polarized toward the tips of small buds and bud necks of larger buds (Figure 1B). Treatment with a-factor, concentrated GFP-A-RAF at the tips of mating projections termed shmoos (Figure 1B). Sucrose density gradient centrifugation was employed to test the observed differences in localization of GFP-RAF proteins. Consistent with the flourescence data, both, C- and -B-RAF segregated with cytosolic proteins in a sucrose density gradient, whereas A-RAF was predominantly found in the heavy membrane fraction (Figure S1A). Taken together, of the three RAF isoforms only A-RAF interacts with yeast membrane components or proteins that are polarized during cell division and mating. Multiple lipid binding motifs of A-RAF are required for its unique localization To test, which A-RAF domain was responsibe for its subcellular distribution in yeast we generated a set of amino- (N-) and carboxy-terminal (C-) A-RAF deletion mutants fused to GFP. The localization of each construct was controlled by fluorescence microscopy. A schematic overview of their subcellular distribution is presented in Figure 1A, right panel. Representative micrographs are shown in Figure 1C. Expression and correct size of the deletion mutants was ascertained by Western blot analysis (Figure S1B). Three types of staining patterns were observed: (i) homogeneous cytosolic as seen with full length B- and C-RAF (see 142–606 construct in Figure 1C), (ii) a punctate pattern (see 88–606 construct in Figure 1C), and (iii) an intermediate pattern, with intensive homogeneous labeling of plasma membranes (see Figure 1C, 1–149 construct as an example). Two different lipid-binding domains have previously been identified in the structure of C-RAF. A phosphatidic acid (PA) binding domain, located in the N-terminal part of CR3 [24,25], and a phosphatidyl serine (PS) binding domain located in the cysteine-rich region of CR1 [25,26]. Moreover, deletion of CR1 of A-RAF (which encompasses RBD+CRD) led to loss of A-RAF- specific binding to PtdIns(4,5)P2 suggesting a third lipid binding site [27]. As these sites are well conserved among RAF isoforms, we mutagenized their key residues, which have previously been shown to affect the interaction of C-RAF with PA and PS (Figure S2) [28]. Substitution of basic residues in the PS site led to localization of mutant protein in the cytosol. This pattern strongly resembled that of N-terminally truncated deletion mutants. In PLoS ONE | www.plosone.org 5 February 2009 | Volume 4 | Issue 2 | e4647