Ineke Keizer-Gunnink

Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting.

We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy. The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway. We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis. Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway. Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy. In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions. Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p. Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated. Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins. Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions.

Essential role of PI3-kinase and phospholipase A2 in Dictyostelium discoideum chemotaxis

Chemotaxis toward different cyclic adenosine monophosphate (cAMP) concentrations was tested in Dictyostelium discoideum cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second-messenger systems PI3-kinase, phospholipase C (PLC), phospholipase A2 (PLA2), and cytosolic Ca2+. The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, whereas both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP3 receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase and insensitive to PLA2 inhibitors. This suggest that PI3-kinase–mediated chemotaxis is regulated by PLC, probably through controlling PIP2 levels and phosphatase and tensin homologue (PTEN) activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca2+.

The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal

Here we describe the identification of the targeting sequence of peroxisomal amine oxidase (AMO) of H. polymorpha. Deletion analysis revealed that essential targeting information is located within the extreme N‐terminal 16 amino acids. Moreover, this sequence can direct a reporter protein to the peroxisomal matrix of H. polymorpha. The N‐terminal 16 amino acids of AMO contain a sequence with strong homology to the conserved PTS2 sequence. Therefore, AMO is considered to be a PTS2 protein.

Characterization of the GbpD-activated Rap1 Pathway Regulating Adhesion and Cell Polarity in Dictyostelium discoideum

The regulation of cell polarity plays an important role in chemotaxis. GbpD, a putative nucleotide exchange factor for small G-proteins of the Ras family, has been implicated in adhesion, cell polarity, and chemotaxis in Dictyostelium. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. These cells overexpressing GbpD are severely impaired in chemotaxis, most likely due to the induction of many protrusions rather than an enhanced adhesion. The GbpD-overexpression phenotype is similar to that of cells overexpressing Rap1. Here we demonstrate that GbpD activates Rap1 both in vivo and in vitro but not any of the five other characterized Ras proteins. In a screen for Rap1 effectors, we overexpressed GbpD in several mutants defective in adhesion or cell polarity and identified Phg2 as Rap1 effector necessary for adhesion, but not cell polarity. Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain.

PI3-kinase signaling contributes to orientation in shallow gradients and enhances speed in steep chemoattractant gradients

Dictyostelium cells that chemotax towards cAMP produce phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] at the leading edge, which has been implicated in actin reorganization and pseudopod extension. However, in the absence of PtdIns(3,4,5)P3 signaling, cells will chemotax via alternative pathways. Here we examined the potential contribution of PtdIns(3,4,5)P3 to chemotaxis of wild-type cells. The results show that steep cAMP gradients (larger than 10% concentration difference across the cell) induce strong PtdIns(3,4,5)P3 patches at the leading edge, which has little effect on the orientation but strongly enhances the speed of the cell. Using a new sensitive method for PtdIns(3,4,5)P3 detection that corrects for the volume of cytosol in pixels at the boundary of the cell, we show that, in shallow cAMP gradient (less than 5% concentration difference across the cell), PtdIns(3,4,5)P3 is still somewhat enriched at the leading edge. Cells lacking PI3-kinase (PI3K) activity exhibit poor chemotaxis in these shallow gradients. Owing to the reduced speed and diminished orientation of the cells in steep and shallow gradients, respectively, cells lacking PtdIns(3,4,5)P3 signaling require two- to six-fold longer times to reach a point source of chemoattractant compared with wild-type cells. These results show that, although PI3K signaling is dispensable for chemotaxis, it gives the wild type an advantage over mutant cells.

Chemoattractants and chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling

During embryonic development, cell movement is orchestrated by a multitude of attractants and repellents. Chemoattractants applied as a gradient, such as cAMP with Dictyostelium discoideum or fMLP with neutrophils, induce the activation of phospholipase C (PLC) and phosphoinositide 3 (PI3)-kinase at the front of the cell, leading to the localized depletion of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) and the accumulation of phosphatidylinositol-3,4,5-trisphosphate (PI[3,4,5]P3). Using D. discoideum, we show that chemorepellent cAMP analogues induce localized inhibition of PLC, thereby reversing the polarity of PI(4,5)P2. This leads to the accumulation of PI(3,4,5)P3 at the rear of the cell, and chemotaxis occurs away from the source. We conclude that a PLC polarity switch controls the response to attractants and repellents.

Dictyostelium chemotaxis: essential Ras activation and accessory signalling pathways for amplification

Central to chemotaxis is the molecular mechanism by which cells exhibit directed movement in shallow gradients of a chemoattractant. We used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signalling module providing activation of Ras at the leading edge, which is sufficient for chemotaxis. The signalling enzymes PI3K, TorC2, PLA2 and sGC are not required for Ras activation and chemotaxis to folate or to steep gradients of cAMP, but they provide a memory of direction and improved orientation of the cell, which together increase the sensitivity about 150‐fold for chemotaxis in shallow cAMP gradients.

Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts

The possible acidic nature of the peroxisomal matrix present in intact yeast cells was studied immunocytochemically, using the weak base DAMP as a probe. Spheroplasts of methanol‐grown Candida boidinii and Hansenula polymorpha were regenerated and incubated with DAMP. After immunogold labelling, using antibodies against DAMP, a specific accumulation of gold particles was observed on the peroxisomal profiles. This labelling was absent in controls, performed in the presence of ionophores or chloroquine. These results support earlier observations, that in intact cells a pH‐gradient exists across the peroxisomal membrane. Experiments, carried out on osmotically swollen spheroplasts indicated that maintenance of this pH‐gradient is strongly related to the cells integrity.

Import of assembled PTS1 proteins into peroxisomes of the yeast Hansenula polymorpha: Yes and No!

Previously, Waterham et al. [EMBO J. 12 (1993) 4785] reported that cytosolic oligomeric alcohol oxidase (AO) is not incorporated into peroxisomes after reassembly of the organelles in the temperature-sensitive peroxisome-deficient mutant pex1-6(ts) of Hansenula polymorpha shifted to permissive growth conditions. Here, we show that the failure to import assembled AO protein is not exemplary for other folded proteins because both an artificial peroxisomal matrix protein, PTS1-tagged GFP (GFP.SKL), and the endogenous dimeric PTS1 protein dihydroxyacetone synthase (DHAS) were imported under identical conditions. In vitro receptor-ligand binding studies using immobilised H. polymorpha Pex5p and crude extracts of methanol-induced pex1-6(ts) cells, showed that AO octamers did not interact with the recombinant PTS1 receptor, at conditions that allowed binding of folded GFP.SKL and dimeric DHAS. This shows that import of oligomeric proteins is not a universal pathway for peroxisomal matrix proteins.

Intramolecular Activation Mechanism of the Dictyostelium LRRK2 Homolog Roco Protein GbpC

GbpC is a large multidomain protein involved in cGMP-mediated chemotaxis in the cellular slime mold Dictyostelium discoideum. GbpC belongs to the Roco family of proteins that often share a central core region, consisting of leucine-rich repeats, a Ras domain (Roc), a Cor domain, and a MAPKKKinase domain. In addition to this core, GbpC contains a RasGEF domain and two cGMP-binding domains. Here, we report on an intramolecular signaling cascade of GbpC. In vitro, the RasGEF domain of GbpC specifically accelerates the GDP/GTP exchange of the Roc domain. Moreover, cGMP binding to GbpC strongly stimulates the binding of GbpC to GTP-agarose, suggesting cGMP-stimulated GDP/GTP exchange at the Roc domain. The function of the protein in vivo was investigated by rescue analysis of the chemotactic defect of gbpC null cells. Mutants that lack a functional guanine exchange factor (GEF), Roc, or kinase domain are inactive in vivo. Together, the results suggest a four-step intramolecular activation mechanism of the Roco protein GbpC: cGMP binding to the cyclic nucleotide-binding domains, activation of the GEF domain, GDP/GTP exchange of Roc, and activation of the MAPKKK domain.