The cytohesin paralog Sec7 of Dictyostelium discoideum is required for phagocytosis and cell motility
- Rolf Müller1,
- Claudia Herr1,
- Salil K Sukumaran1,
- Napoleon Nosa Omosigho1,
- Markus Plomann2,
- Tanja Y Riyahi1Email author,
- Maria Stumpf1,
- Karthic Swaminathan1,
- Marios Tsangarides1,
- Kyriacos Yiannakou1,
- Rosemarie Blau-Wasser1,
- Christoph Gallinger3,
- Michael Schleicher3,
- Waldemar Kolanus4 and
- Angelika A Noegel1Email author
© Müller et al.; licensee BioMed Central Ltd. 2013
Received: 21 January 2013
Accepted: 29 July 2013
Published: 1 August 2013
Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration.
We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis.
Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton.
KeywordsARFGEF Cell adhesion Cell migration Phagocytosis Phosphoinositide binding
ADP ribosylation factor (ARF) GTPases have roles in vesicular transport, in the regulation of actin cytoskeleton dynamics, cell adhesion, cell migration and in signal transduction processes. They depend on specific nucleotide exchange factors, the ARFGEFs, which through their conserved Sec7 domain (Sec7d) catalyze the GDP to GTP exchange. Six subfamilies of ARFGEFs exist in eukaryotes, the large ARFGEFs of the GBF and BIG family, and the small ARFGEFs of the cytohesin, EFA6, BRAG and FBX family. For all ARFGEFs localization to membranes is important for their functions in ARF activation. BIG1 and BIG2 localize to the TGN and endosomes, GBF localizes to the Golgi, and cytohesins are found at the cell periphery where they function in plasma membrane endosomal membrane trafficking and in signal transduction pathways .
Cytohesins are composed of a Sec7d domain and a pleckstrin homology (PH) domain of the cytohesin type followed by a polybasic stretch which together with the PH domain is necessary for plasma membrane association and biological function . The PH domain of the cytohesins specifically binds to phosphoinositides, whose levels are influenced by PI3-kinases in response to upstream signals. Upon recruitment to the plasma membrane cytohesins activate ARFs, which influence the cortical actin cytoskeleton, endocytic processes and phagocytosis. Cytohesins also regulate signaling pathways by functioning in scaffolding complexes. Such a complex was reported for cytohesin-2 which through its N-terminal sequences could assemble a protein complex that contained the RacGEF DOCK180 and promoted Rac activation and cell migration . Furthermore, in dendritic cells cytohesin-1 regulated migration in vivo by functioning upstream of RhoA activation .
The lower eukaryote D. discoideum harbors six ARFGEFs belonging to three families, the GBF and BIG family with one and two representatives each, and three members of the cytohesin family harboring the characteristic Sec7-PH tandem . The finding of this class of proteins in a lower eukaryote was surprising as cytohesins had been reported only for metazoans . Instead of the typical coiled coil domain at the N-terminus, the Dictyostelium cytohesins have variable domains. SecG (DDB_G0287459) has several ankyrin repeats and DDB0233591 (DDB_G0279241) harbors four predicted transmembrane domains whereas the N-terminus of DDB0233617 (DDB_G0272486) which we designate Sec7 has no putative conserved domains . Instead, homopolymer tracts of asparagine and threonine are present. Such homopolymer tracts are quite frequent in D. discoideum proteins [7, 8].
In previous work we had analyzed the function of SecG and found that mutant cells lacking SecG had reduced cell-substratum adhesion whereas cell cell adhesion was not affected. In cell migration analysis speed was significantly reduced, persistence and directionality of migration were unaltered. Here we analyze the Sec7 protein and characterize Sec7 deficient cells. We find that Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P3. We compare these data with the one reported for known PH sensors and ArfA, the single ARF GTPase of D. discoideum.
Sec7 associates with the plasma membrane
The PH domains of cytohesins bind polyphosphoinositides and have different affinities and specificities for PI(3,4)P2, PI(4,5)P2 and PI(3,4,5)P3 depending on their structure [2, 13, 14]. The signaling lipids PI(3,4)P2 and PI(3,4,5)P3 are rare components of the plasma membrane and are produced in response to a stimulus which then recruits the PH domain proteins to the membrane. PH domains have a conserved core fold consisting of a seven strand β-barrel followed by an α-helix [11, 15]. The PH domain of Sec7 has these structural elements too, however, when we modeled the Sec7 sequence to the crystal structure of the autoinhibited form of Grp1 Arf GTPase Exchange Factor we identified a nine strand β-barrel (Figure 1B). The signature motif for 3-phosphoinositide binding K Xm KxR Xn Y  is modified to I X10 SxK X10 F (m = 5-10; n = 6-13). Changes in these positions are also present in PH domains of other proteins (Figure 1C) . A recently described glutamate in the PH domain of cytohesin-3 (GRP1), a sentry glutamate, appears to be essential for specific PI(3,4,5)P3 binding by the cytohesins as a charge reversal by mutation to lysine (E345K) enhanced the affinity for PI(4,5)P2 and yielded constitutive plasma membrane binding . In Sec7 the glutamate is replaced by a glutamine which is considered a neutral residue (Figure 1A,B). A phylogenetic analysis of the Sec7 PH domain placed the D. discoideum protein close to GRP1 proteins of several species including the human protein (Additional file 1: Figure S1).
A polybasic domain which follows the PH domains in all cytohesins is not present in Sec7. However, the C-terminus of Sec7 encompassing the PH domain and the remaining stretch of amino acids is highly basic with a pI of 9.58 which is presumably due to two poly asparagine stretches and an abundance of lysine residues near the C-terminus (10 lysine residues out of 56 residues).
In cell fractionation experiments the protein was predominantly found in the cytosolic fraction which is in agreement with data obtained for cytohesins. A signal in the 100,000 × g pellet was only obtained when we loaded the material from 2×107 cells/ml whereas for whole cell lysate and 10,000 × g pellet and supernatant, respectively, the proteins represent the material from 2×105 cells/ml (Additional file 2: Figure S2A). We also expressed GFP-tagged Sec7 domain (residues 256-443) and found that it was present throughout the cytosol and did not relocate to the plasma membrane (data not shown).
Identification of potential partners of Sec7
Potential binding partners of Sec7 and its domains
Identified in Sec7 domain pull down
Identified in PH domain pull down (C-terminus)
Identified in GFP-Sec7 immuno-precipitation
Identified in N-domain pull down
Membrane trafficking, membrane associated processes
cog3, oligomeric Golgi complex component
osbH, oxysterol binding
Present in macropinocytic proteome; conserved; transporter?
nsfA, N-ethylmaleimide-sensitive fusion protein
p3d1, delta adaptin (endosomal membrane)
Ap1b1, adaptor-related protein complex 1, beta 1 subunit, beta adaptin, highly similar to AP-1 complex subunit beta-1 (AP1B1) and AP-2 complex subunit beta-1 (AP2B1), which play a role in clathrin-dependent protein sorting
tgrO4, immunoglobulin E-set domain-containing protein, tgr (tiger) = Transmembrane, IPT, IG, E-set, Repeat protein
copG, adaptin N-terminal domain-containing protein
coatomer protein complex gamma subunit
Single C-terminal TM
Dnajc13, DnaJ (Hsp40) homolog, subfamily C, member 13, very similar to the mammalian DnaJ homolog subfamily C member 13, required in D. melanogaster (Rme-8) for receptor-mediated endocytosis 8
myoI, class VII unconventional myosin
myosin VII, similar to the conserved MYO7A; unconventional myosin required in Dictyostelium for phagocytosis and substrate adhesion, interacts with talA
vps13A, vacuolar protein sorting-associated protein 13 family protein, putative ortholog of S. cerevisiae VPS13, involved in vacuolar protein sorting and protein-Golgi retention
Related to signal transduction
pppA, protein phosphatase 2A subunit A
GTP-binding protein, 92 kDa
zizB, DOCK family protein, putative guanine nucleotide exchange factor (GEF)
zizA, DOCK family protein, putative guanine nucleotide exchange factor (GEF)
Centrosomal, 80 kDa
cytoplasmic dynein heavy chain,
dynein beta chain, flagellar outer arm
myoI class VII unconventional myosin
myosin VII, similar to the conserved MYO7A; unconventional myosin required in Dictyostelium for phagocytosis and substrate adhesion, interacts with talA
Characterization of a D. Discoideum Sec7 deficient mutant
For the generation of Sec7 deficient cells (sec7 - ) we used a gene replacement vector which contained nucleotides 1 to 441 and 1981 to 2469 of the cDNA. The intervening gene sequence was replaced by a blasticidin resistance cassette. Successful integration of the vector into the genome was confirmed by PCR and Southern blot analysis (Figure 4B,C). Several independent transformants were isolated, the characterization of one of them is shown. In the subsequent analysis we focused primarily on properties of the cells that are associated with processes involving regulation of the actin cytoskeleton and membrane dynamics.
Growth on a lawn of Klebsiella as determined by measuring the increase in diameter of the colony was significantly reduced (Figure 5B). Such a behavior could be due to reduced phagocytosis or to altered motility. Hence we assayed the phagocytic capability following yeast particle uptake and found that fewer sec7 - cells had ingested one or more yeast particles after 45 min than AX2 cells. A quantitative evaluation showed that significantly fewer sec7 - cells (~79%) had taken up yeast cells as compared to AX2. sec7 - cells expressing GFP-Sec7 reached almost wild type level with ~95% (Additional file 4: Figure S3). The failure to completely restore wild-type behavior may lie in different expression levels and partial loss of expression.
Cell substrate and cell cell adhesion are two characteristics which are potentially influenced by the cytohesins as they affect events at the plasma membrane such as integrin activation . Consequently we wanted to assess the role of Sec7 for these two aspects. Cell substrate adhesion is assayed by subjecting cells that have attached to a plastic surface to a rotation on an orbital shaker at 100 rpm. The percentage of detached cells after one hour was similar for AX2 and mutant cells. Upon increase of rotation to 160 rpm the results for wild type and mutant showed no significant difference either (data not shown). Cell cell adhesion was tested in developing cells. During development D. discoideum cells express specific cell surface proteins like contact site A which mediate cell adhesion and allow aggregate formation . When we assayed aggregate formation during development in shaking suspension by following the decrease in optical density of the suspension we did not detect differences between wild type and mutant (data not shown). This was also taken as an indication for correct expression of the cell adhesion molecules and for normal development of the mutant.
Later developmental stages were assayed by depositing cells on phosphate agar plates for starvation. The sec7 - strains formed aggregates, mounds, slugs and fruiting bodies in a timely fashion. Furthermore, the fruiting bodies had comparable morphologies (data not shown).
Slugs are phototactic and migrate towards light. Phototaxis is an essential feature in the wild where Dictyostelium has to reach the soil surface from where the spores can be dispersed. Directed migration of slugs towards light was tested by keeping the plates in the dark and providing a lateral light source. After incubation for two days the trails of the slugs were stained with Amido Black and the migration pattern and the light sensing evaluated. AX2 slugs migrated over long distances and almost directly towards the light source. Many of the slugs had reached the edge of the plate. sec7 - slugs had a phototaxis defect. Their migration trails were shorter and the slugs never reached the edge of the plate. Moreover, their directionality was altered and the angle of deviation during slug phototaxis was increased. AX2 slugs migrate with an angle of ~13 degrees, sec7 - slugs with an angle of ~55 degrees and for sec7 - slugs expressing Sec7-GFP the angle was reduced to ~31 degrees (Figure 5C). We conclude that sec7 - slugs can sense light, but directionality is impaired.
Ability of sec7 - cells to secrete enzymes during early development: secretion of α-Mannosidase
% secreted enzyme after
% total enzyme activity after 6 hours
Ability of sec7 - cells to secrete enzymes during early development: total mannosidase activity in wild type and mutant cells at various stages of development
Ability of sec7 - cells to secrete enzymes during early development: phosphodiesterase production
PDE activity (U/ml) determined at the indicated time points after begin of starvation
sec7 - (t1)
sec7 - (t2)
sec7 - (t3)
Analysis of signaling to the actin cytoskeleton, cell motility and chemotactic behaviour
Cell motility of aggregation stage cells
Direction change (deg)
Summary of phenotypes observed in the sec7 - strain
Growth in axenic medium (measure of macropinocytosis)
Growth on E. coli in shaken suspension
Growth on a lawn of Klebsiella
Phagocytosis of yeast cells
uptake reduced to 60% of AX2 level
Cell substrate adhesion
Cell cell adhesion
directionality impaired, slug trail length reduced
Localization of F-actin and actin associated proteins (actin, CAP)
Integrity of ER membrane system (PDI staining)
Secretion of α-mannosidase
secretion occurs normally, however enzyme levels are reduced (~76% of AX2 level)
Secretion of phosphodiesterase
secretion appears to be impaired, enzyme levels are reduced (~50% of AX2 level)
F-actin assembly after cAMP stimulation
Persistence during migration
Members of the cytohesin family of GEFs are recruited to the plasma membrane based on the affinity of their PH domain for specific phosphatidylinositol phosphates. A further mechanism is through interaction with the GTP bound form of ARF6 and ARL4 which leads to cytohesin recruitment and activation of ARF6 and ARF1 [32–34]. The activation of ARF proteins stimulates signaling pathways that regulate membrane trafficking and cell motility. Mammals have six ARFs forming three classes and playing roles in different cellular pathways . D. discoideum harbors a single ARF homolog encoded by the arfA gene on chromosome 5. This is similar to Giardia lamblia, a flagellated protozoan parasite, which also has one ARF only. ArfA (DDB0191101) is most closely related to ARF1 with 85% identical and 90% homologous residues when compared to human ARF1 (NP_001019397.1) (E value, 3e-86) and less related to human ARF6 (CAG46762.1) sharing 67% identical and 82% homologous residues (E value 1e-69). In addition to ArfA D. discoideum has ten ARF related proteins, ArrA–K. ArfA is present in a macropinocytic proteome  whereas this has not been reported for Sec7. ArfA has also been studied by Chen et al.  who showed that two ArfGAPs belonging to the ASAP/ACAP type of ArfGTPases, which are distinguished by BAR, PH and Ank domains, act on ArfA. The D. discoideum ArfGTPases have roles in actin cytoskeleton organization and spore production. The studies on ACAP were more recently extended by Dias et al.  who revealed additional roles in cytokinesis, cell migration and cytoskeleton dynamics.
Another ArfA binding protein in D. discoideum is AdcA. This arrestin related protein bound ArfA in its GDP-bound conformation . AdcA was associated with the endocytic pathway and early endosomes, and faintly labeled the plasma membrane. GFP-tagged ArfA localized in the cytosol, at the plasma membrane and mainly in the perinuclear region at the Golgi apparatus. From in vivo studies the authors concluded that ArfA-GFP was present on vesicles and tubules moving towards and away from the Golgi apparatus. They proposed a role for ArfA in trafficking events that link the Golgi to other organelles such as endosomes. Although ArfA had been found in proteomic studies on phagosomes and macropinosomes [36, 40], ArfA-GFP was not convincingly located at these structures in vivo which was thought to be due to transient interactions. The absence of GFP-Sec7 from these locations in our studies is compatible with these results.
In our attempt to identify potential binding partners of Sec7 we carried out pull downs using domains of Sec7 expressed as GST fusion proteins in E. coli and full length protein expressed as GFP fusion in D. discoideum. The majority of the putative interaction partners from all pull downs belonged to the processes of trafficking and vesicle trafficking although the overlap between the individual pull downs was limited. This could be due to the conformations of the proteins and the different accessibilities of the sequences. For some of the putative interaction partners mutants are available and their phenotypes can be compared to the one of the sec7 - mutant. We did see overlaps of the phenotypes in several cases which might indicate that the proteins act in the same pathway. This applies particularly to phagocytosis and chemotactic motility (Additional file 3: Table S2).
Phosphoinositides are tightly regulated during chemotaxis in D. discoideum, in particular, PI(3,4,5)P3 gradients are formed within the plasma membrane. They are thought to be of differing importance for sensing of shallow and steep cAMP gradients [30, 41]. The PH domain of the cytohesin family of ARF-GEFs can act as PI(3,4,5)P3 sensor. We found that D. discoideum Sec7 had highest affinity for this phosphoinositide in lipid overlay assays followed by PI(4,5)P2, whereas in liposome binding assays it preferred PI(4,5)P2 and PI(3,4)P2 over PI(3,4,5)P3. When we analyzed GFP-tagged Sec7 in vivo the protein decorated the plasma membrane and it did not visibly associate with those membrane regions where PI(3,4,5)P3 formation is thought to occur. The group of C. Weijer  had used a panel of PH domains with specificities for PI(3,4,5)P3 and PI(3,4)P2, among them the PI(3,4,5)P3 specific PH domain of GRP1 (cytohesin-3), with which they analyzed the formation of these signaling molecules during phagocytosis and chemotaxis. They concluded that PI(3,4,5)P3 levels transiently increased during phagocytosis and macropinosome formation at sites of engulfment as revealed by the recruitment of GRP1-PH to these regions. During chemotaxis towards cAMP PI(3,4,5)P3 was formed and degraded to PI(4,5)P2 in the plasma membrane. GRP1-PH and CRAC-PH, a D. discoideum protein specific for PI(3,4,5)P3, translocated to the plasma membrane following cAMP stimulation . Interestingly, the Weijer group found that different PI(3,4,5)P3 binding PH domains behaved differently as GRP1-PH exhibited maximum binding several seconds later than other PH domains and remained at the membrane much longer which might be indicative of further determinants.
GFP-Sec7 did not exhibit a comparable pattern of plasma membrane association. Instead, it was constitutively associated with the plasma membrane and consistently disappeared from regions forming a new pseudopod, undergoing phagocytosis or macropinocytosis which require the insertion of new membrane. Sec7-GFP showed a behavior complementary to the one of PH domains sensing PI(3,4,5)P3 and rather resembled the pattern reported for the phosphatase PTEN and the PH domain of PLCδ . Like Sec7 deficient cells the PTEN null cells showed an impairment in phagocytosis of yeast cells and a normal uptake of bacteria . They also had a cell migration defect [43, 44].
The analysis of a Dictyostelium Sec7 mutant implicates the protein in processes that are related to membrane flow and actin dynamics and reveals a conserved function for this class of proteins. Its PH domain has the potential to recognize PI(4,5)P2 and to a lesser extent PI(3,4,5)P3 in vitro. The protein associates with the plasma membrane, however, during macropinocytosis, phagocytosis and pseudopod extension it is lost. It therefore does not act as a sensor for 3-phosphoinositide dynamics. This is supported by the absence of the sentry glutamate in Sec7 which in the cytohesins is essential for specific plasma membrane targeting. Instead, the glutamate is replaced by glutamine. The Sec7 plasma membrane association is presumably mediated by PI(4,5)P2 binding, however, other determinants such as binding to interacting proteins might also be important factors.
Materials and methods
Growth and development of D. Discoideum strains and mutant generation
D. discoideum strains used were AX2, a Sec7 deficient strain derived from AX2 (sec7 - ), and sec7 - expressing GFP-Sec7. They were grown in shaking suspension (160 rpm) in axenic medium at 22ºC or on a lawn of Klebsiella on SM agar plates . For growth on E. coli in shaking suspension (160 rpm) E. coli B/r was harvested and resuspended in Soerensen phosphate buffer (17 mM sodium-potassium-phosphate, pH 6.0) at a density of 1010 cells/ml. Inoculation was with 5×105D. discoideum cells/ml. Growth was determined by following the increase in cell number over time. Development was done with cells starved in Soerensen phosphate buffer at a density of 1×107 cells/ml in shaking suspension. Under these conditions, development proceeded until the tight aggregate stage. Upon development on a solid substratum fruiting body formation occurred. For this 5×107 cells were spread onto phosphate agar plates (10 cm in diameter) and incubated at 22ºC until fruiting bodies had formed. For evaluation of slug migration and phototaxis 5×105 cells in 5 μl Soerensen phosphate buffer were spotted in the center of a water agar plate. Incubation was in the dark with a lateral light source. After 48 hours cells had formed slugs which migrated towards the light. They were transferred to nitrocellulose filters and detected by staining with Amido Black (0.1% in 25% isopropanol and 10% acidic acid).
The Sec7 cDNA was amplified from cDNA that had been prepared from strain AX2, cloned into pGEM-T Easy (Promega). The sequence was verified and used for all further cloning steps. For inactivation of the Sec7 gene, a gene replacement vector was generated. Residues 4 to 441 and 1981 to 2469 of the cDNA were cloned into pGEM-T Easy carrying a Blasticidin resistance gene under the control of the actin 15 promoter . The plasmid was transformed into AX2 and transformants were selected using Blasticidin S (MP Biomedicals, Eschwege, Germany) at 1.5 μg/ml. Single colonies were selected on a Klebsiella lawn, DNA was isolated from nuclei using phenol/chloroform extraction  and PCR analysis was carried out with primers that revealed the gene replacement event. The gene replacement was further confirmed by Southern blot analysis. Several independent clones were identified, two of them were further analysed. As the results did not differ, the characterization of one of the clones is presented in the Results section.
Expression of recombinant protein
For expression of recombinant Sec7 polypeptides as glutathione S transferase (GST) fusion proteins in E. coli, cDNA fragments encoding the N-terminal domain (amino acid residues 1-382), the Sec7 domain (amino acid residues 256-443) and the C-terminus encompassing the PH domain (amino acid residues 381-931) and a polypeptide containing only the PH domain (amino acid residues 454-577) were cloned into pGEX vectors (GE Healthcare Life Sciences). E. coli strains XL1 Blue and, in case of the Sec7 domain, Arctic Express Ril (Agilent Technologies) were used for expression of the GST fusion proteins. Full length Sec7 cDNA was cloned into pDex79 and expressed as GFP fusion (GFP-Sec7) under control of the actin 15 promoter . GFP was fused to the N-terminus of Sec7. The plasmid was transformed into sec7 - cells. Selection was with G418 (Life Technologies Corporation) at 4 μg/ml. For expression of the Sec7 domain, cDNA sequences corresponding to amino acid residues 252–443 were cloned into pDex79.
Pull down assays and immunoprecipitation
To identify interaction partners of Sec7, GST-Sec7 N-terminus, GST-Sec7 domain, GST-Sec7 C-terminus and GST for control were bound to glutathione sepharose 4B beads (GE Healthcare) and used for pull down assays. Incubation with cell lysates varied from 2 hours to overnight and was performed at 4ºC. Lysates were prepared from AX2 cells, sec7 - expressing Sec7-GFP and sec7 - cells expressing LimD-GFP  using the following buffer: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP 40, 0.5 mM EDTA, 1 mM PMSF and protease inhibitors (Sigma). Lysis was controlled microscopically. For immunoprecipitation of GFP-Sec7, monoclonal antibodies (mAb K3-184-2) as well as polyclonal antibodies against GFP bound to protein A sepharose beads were used . Protein A sepharose beads were used as control to exclude proteins that bind to the sepharose matrix. The proteins from pull downs, immunoprecipitations and controls were separated by SDS-PAGE (10% to 12% acrylamide), stained with Coomassie Blue, bands from control and experiment were cut out and proteins processed for LC-MS at the Bioanalytics Facility of the CMMC. The Mascot search engine was used for identification of the proteins. For verification of the interaction immunoprecipitation of GFP-Sec7 was repeated and probed for the presence of coronin and subunit α-4 (psmA4, (DDB0214953)) of the 20S proteasome using mAb 176-3-6 and 159-83-10, respectively [48, 49].
Growth analysis, uptake of yeast particles and adhesion assays were done as described , analysis of F-actin assembly after cAMP stimulation was done as described . Mannosidase activity in cell pellets and in the supernatant was determined according to Loomis . In brief, cells were starved at a density of 1×107 cells/ml. At the beginning of the experiment (t0) and after 2, 4 and 6 hours 500 μl were taken to measure mannosidase activity. For determination of secreted enzyme 100 μl of the supernatant were mixed with 100 μl Na-citrate buffer (pH 5.0) and 200 μl substrate solution (2 μl p-nitrophenyl-α-D mannopyranoside (150 mM). The substrate was dissolved in DMF. The reaction was stopped after 30 min incubation at 37ºC by addition of 600 μl sodium borate (0.2 M, pH 9.8) and the product extracted into butanol. Nitrophenol formation was estimated by measuring the absorbance at 405 nm. For determination of total enzyme activity cells were lysed by addition of Triton X-100 (0.5%). cAMP phosphodiesterase (PDE) activity was determined using a coupled enzymatic assay with cAMP as substrate . AMP, the product of the hydrolysis, was further converted to IMP by adenosine deaminase. IMP was then converted to inosine by alkaline phosphatase. The decrease of the absorption at 265 nm was a measure of inosine formation which in contrast to adenosine does not absorb light at 265 nm. Reagents and enzymes were from Sigma. For immunofluorescence analysis methanol fixed cells were stained for actin (mAb act1 ), CAP (mAb 223–445-1 ), protein disulfide isomerase (PDI, mAb 221-135-1 ) and annexin 7 (mAb 185-338-1 ). For detection goat anti mouse antibodies coupled to Alexa Fluor 488 (Life technologies) were used. Analysis of fixed and living cells was done by laser scanning confocal microscopy using a Leica TCS SP5 microscope.
For cell motility analysis cells were plated after ~ 6 hours of starvation in a chamber (ibidi GmbH-Martinsried, Germany) and migration towards aggregation centers or towards a micropipette filled with 10 μm cAMP was followed. Analysis was carried out by using the DIAS system as described . For phototaxis 5×105 cells are placed in the center of a water agar plate. Slugs were allowed to form and migrate towards light. After 48 h, slugs and slime trails were transferred to nitrocellulose filters and stained with Amido Black.
Liposome binding assay
Phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3 were obtained from Sigma and diluted in chloroform. Liposome binding experiments were performed with a modified published liposome binding assay protocol . Lipid mixtures containing 65% PC, 20% PE, 5% PS and 10% individual phosphoinositides were produced by mixing appropriate lipid solutions in chloroform/methanol. Slow flow nitrogen gas was used for the production of a film on the glass and vacuum desiccation for 30 min for solvent removal. Sterile-filtered sucrose binding buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 1 mM EDTA and 0.1 M sucrose) was added to a final lipid concentration of 1mg/ml and incubated at 37ºC for 2 hrs. Lipids were then sonicated in a waterbath-sonicator for 10 sec.
To test liposome binding, a 100 μl reaction mixture of freshly prepared liposomes and 5 μg of purified protein were incubated for 15 min at room temperature and centrifuged at 100,000 × g (42,000 rpm) at 4ºC for 25 min in a Beckman table top ultracentrifuge Optima TLX (TLA 45 rotor). The supernatant was saved, and the pellet was resuspended in 100 μl of sucrose binding buffer. Both fractions were then analyzed by SDS-PAGE followed by Coomassie blue staining. ImageJ was used for quantification.
Phosphoinositide-binding assays using lipid strips supplied by Echelon Biosciences, Inc. (Salt Lake City, Utah, USA) were performed as described . For statistical analysis the Student's t test was used. For cell fractionation cells were lysed using Nuclepore filters (Whatman) in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl and protease inhibitors (Sigma). Sequential centrifugation steps were done at 400 × g (2 min) to remove unlysed cells, 10,000 × g (10 min) to pellet nuclei and 100,000 × g (60 min) to separate membrane and cytosolic fractions. Proteins were separated on SDS-PA gels (10% acrylamide), blotted onto nitrocellulose membranes and GFP-tagged protein detected with mAb K3-184-2 and for a cytosolic control protein using enhanced chemiluminescence . Total RNA was isolated using phenol extraction. Quantitative Real Time PCR was done as described .
Work in the authors’ labs (AAN and WK) is supported by the DFG, SFB 670 and 832, respectively, MS by SFB 863 and 914. TR is supported by SFB 670, SKS is a member of the CECAD graduate school. We thank Berthold Gaßen for antibody generation, Debora Hofmann and Philipp Niehues for help with immunofluorescence analysis, and Dr. S. Müller (CMMC) for performing mass spectrometry analysis.
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