DYNAMICS OF LYSOSOMAL COMPARTMENTS: MEMBRANE FUSION, MEMBRANE FISSION AND OSMOADAPTATION
We study vesicular transport in eukaryotic cells, focusing on the question of how intracellular membranes fuse and fragment and how these processes are coordinated to regulate copy number and size of lysosomal compartments. Furthermore, we study the synthesis and degradation of polyphosphate in the lysosome-like vacuole. Polyphosphates are intimately linked to osmoregulation in many cells, e.g. also in parasites, where they are necessary for the functioning of contractile vacuoles and parasite survival.
1. MEMBRANE FUSION
Membrane fusion is required for vesicular traffic of proteins and lipids between organelles. In exocytosis it controls many vital processes, such as signal transduction among neurons, secretion of hormones or digestive enzymes, the abundance of sugar transporters, or the cytotoxic activity of T-lymphocytes. Also uptake and persistence of intracellular parasites are closely related to vesicular traffic.
Membrane fusion reactions on compartments of the secretory and endocytic pathway share conserved factors. Fusion requires SNAREs which occur in compartment-specific cognate combinations of vesicular (v-) and target (t-) SNAREs. V- and t-SNAREs can form trans-complexes between the two fusion partners which may hold the membranes in close apposition (Fig. 1). We explore how this state of membrane docking evolves into lipid flux and fusion pore opening and how the fusion machine is connected to signal transduction cascades regulating membrane traffic in response to external stimuli. Both issues are studied in a cell-free system reconstituting fusion with purified organelles.
To this end we have established assays that allow us to separately measure the transition of lipids from the outer and inner membrane leaflets and to measure the opening of fusion pores that lead to mixing of vesicle contents (Reese et al., 2005). We try to combine these biochemical and spectroscopic approaches with electrophysiology in order to explore the dynamics of the fusion pore in detail and correlate its behaviour with genetic manipulations of the fusion proteins.
Our model system: Homotypic vacuole fusion
Vacuoles have properties similar to lysosomes, because they are responsible for hydrolytic degradation in yeast cells, but also to acidocalcisomes, because the are essential for osmoregulation and ion homeostasis. Yeast vacuoles fragment and fuse in the course of the cell cycle and in response to changes in osmotic pressure or nutrient availability. Vacuoles are a great model system to study membrane dynamics since they allow to combine the facile manipulation of a genetically accessible organism with the analytical power of a cell-free system. This enables us to develop experiments that could not be performed in higher eukaryotic cells with comparable ease and resolution.
Many factors and events defined by our work on vacuole fusion also have roles in other fusion reactions: Ca2+ release from the organellar lumen and for calmodulin have also been implicated in fusion at the Golgi, at mammalian endosomes and at lysosomes. Protein phosphatase I controls also fusion at the Golgi and the VTC proteins also influence ER-Golgi trafficking. Finally, work by several other groups confirmed that the V0 sector of the V-type H+-ATPase, which is necessary to induce lipid flux in vacuole fusion, also stimulates exocytosis of neurotransmitters, insulin, and multivesicular bodies, and that it is required for phagosome-lysosome fusion. Studying vacuole fusion hence elucidates general aspects of membrane traffic which are also relevant to vesicular transport in higher eukaryotic cells.
2. CONTROL OF ORGANELLE NUMBER AND SIZE BY MEMBRANE FUSION AND FISSION
Vesicular traffic consists of cycles of membrane fission (vesicle formation) and membrane fusion (vesicle consumption). Similarly, many organelles exist in an equilibrium of fragmentation into smaller units (e.g. during mitosis) and fusion into larger structures. Prominent examples are the Golgi, the nuclear envelope, mitochondria, vacuoles and lysosomes. How are size, copy number and shape of an organelle determined and how are the elementary processes of membrane fission and fusion coordinated in order to control these parameters? We study this evident but poorly explored problem. Our favourite organelle, yeast vacuoles (lysosomes) are an excellent model system for this question: Vacuoles change copy number and size in the cell cycle and upon shifts of media; due to their large diameter (up to 5 µm) these changes can be assayed by fluorescence microscopy and are amenable to genetic screening. Moreover, an in vitro system for vacuole fusion exists and we succeeded in reconstituting also cell-free vacuole fission with purified organelles.
We combine several approaches to build an experimental toolkit for vacuole fission and to characterize this reaction in detail: (1) Identification of fission proteins by mutant screening. To this end we exploit the fact that active vacuole fragmentation can be induced by salt treatments of the cells (Fig. 3). (2) Reconstituting fission in vitro (Fig. 4) and developing methods to quantitate it. (3) Time-resolved confocal fluorescence microscopy of fission proteins in vivo and in vitro. (4) Biochemical characterization of fission protein associations and their changes during fission. With these approaches we hope to identify the vacuolar fission apparatus and elucidate its functioning.
In a second line of experiments we explore how the fission apparatus physically and functionally interacts with the – already well-defined – vacuolar membrane fusion machinery. We characterize the impact of cell cycle regulators and signaling pathways on these interactions. These studies may elucidate how membrane fission and fusion components are coordinated to control size and copy number of an organelle (Peters et al., 2004; Baars et al., 2007).
3. THE ROLE OF POLYPHOSPHATES IN EUKARYOTES: OSMOREGULATION, PHOSPHATE STORAGE AND THE VIRULENCE OF PARASITES
Inorganic polyphosphate (polyP) is a polymer of dozens to hundreds of orthophosphate (Pi) units linked by energy-rich phosphoanhydride bonds. PolyP occurs in all life forms. In prokaryotes it is a store of phosphate and energy and augments stress resistance. Large quantities of polyP that are synthesized by eukaryotic plankton form marine sediments and influence the global phosphorous cycle. PolyP is important in plant nutrition by mycorrhizal symbioses, participates in calcification of bones in mammals, and is crucial for osmoregulation via contractile vacuoles and acidocalcisomes in parasites such as Leishmania, Plasmodia and Trypanosomes. The identity of a eukaryotic polyP-synthesizing enzyme has long been elusive.
Fig. 4: Two representations of the catalytic domain of the yeast polyP polymerase bound to polyP (yellow/red) showing secondary structure (left) and electrostatic potential (right)
Our structural and biochemical analyses have identified this enzyme as a domain of a large vacuolar membrane protein complex. Several crystal structures revealed that this eukaryotic polyP polymerase is completely distinct from bacterial enzymes and shows novel mechanisms of nucleotide binding and turnover (Hothorn et al., Science, in press). We hypothesize that the complex carrying the polyP polymerase activity may not only synthesize polyP at the cytosolic face of the vacuolar membrane but at the same time form a channel that translocates the nascent chain across the membrane into the vacuoles. We now develop assays to measure polyphosphate synthesis in real time and to purify the complex and reconstitute it in liposomes in order to show its function as a polyphosphate translocase. In addition, we try to determine the structure of the whole complex, including its transmembrane parts and the polyphosphate chain. If successful, this might reveal a completely novel type of coupled enzyme/translocase.
The biology of polyphosphates is virtually unexplored territory in eukaryotes. Since most critical residues are in the catalytic center of the yeast polyphosphate polymerase are conserved we can use our structural and mechanistic information from the yeast enzyme to explore the consequences of shutting down polyphosphate synthesis in other organisms. This concerns e.g. their role in unicellular eukaryotes, among them parasites causing malaria, sleep sickness and leishmaniasis. These organisms concentrate polyphosphates in vacuoles and acidocalcisomes. Here, polyP is assumed to participate in osmoregulation, e.g. by driving the pumping cycles of their contractile vacuoles via rapid polyP hydrolysis and re-synthesis. These issues not only touch fundamental questions of eukaryotic cell biology but offer also interesting biomedical perspectives.