An example
This video shows an example of membrane fusion. First, two organelle membranes adhere to one another, a process called docking. They then fuse into one continuous membrane. Here, we’re looking at a cell-free system: the fusion of lysosomal vacuoles, purified from yeast cells. Insights obtained from studies of yeast vacuole fusion have turned out to have broader significance for our understanding of many other fusion events, including the exocytosis events that mediate neurotransmission. Vacuole docking and fusion, like most other membrane fusion events, is regulated by switching elements called Rab small G-proteins and catalyzed by SNARE proteins. A third family of protein machines, the Vps-C complexes, link Rab signaling to SNARE function on endosomes and lysosomes. Additional factors including kinases add layers of regulation to the system, and it is likely that fifty or more proteins are involved in just this one fusion event.
The objective
Our group’s overall goal is to understand how the complex membrane-enclosed organelles of eukaryotic cells are constructed and modified during differentiation, organ development, and disease. Our focus is to understand how the endolysosomal pathway is regulated, in molecular detail. More than ten thousand different proteins are encoded by the human genome; about a third of these reside in membranes. Newly synthesized membrane and secreted proteins are carried through the secretory pathway, while membrane proteins and endocytosed materials marked for destruction travel down the endocytic pathway to meet their fate in the hydrolytic lysosome. In addition to endocytic traffic, the lysosome is the terminal destination of cytoplasmic material taken up through the autophagy pathway. The lysosome therefore receives cargo from the extracellular environment, from the cell’s plasma membrane boundary, and from the cytoplasm.
A bit of background
We are currently working to understand fundamental mechanisms of membrane traffic into and among the endolysosomal organelles of budding yeast, Saccharomyces cerevisiae. This model organism is unparalleled for studies of fundamental, conserved mechanisms of membrane traffic. It offers simple propagation, unequalled genetics and functional genomics, and superb biochemical assays of membrane fusion and trafficking. We are problem-driven and employ diverse approaches including genetics, cell-free fusion reactions, protein biochemistry, chemically defined reconstitution systems, and optical techniques including high-sensitivity microscopy. Through collaborations we employ electron microscopy (tomography, single particle analysis), X-ray crystallography, mass spectrometry, and high-throughput functional genomic screening.
Eli Metchnikov discovered the endocytic system and recognized its central role in immunity a century ago. The endocytic system also has crucial roles in growth, development, cell transformation in cancer, and fat and cholesterol metabolism. Secretory and endocytic organelles are maintained dynamically though cycles of cargo selection, carrier vesicle budding, and vesicle docking and fusion with target membranes. Work by many scientists has yielded a catalog of proteins and protein interactions that direct and execute these processes, but many fundamental questions are unanswered.
Projects
Recently, we have made substantial progress in three areas. In the first two projects, we are studying the mechanisms of membrane tethering and fusion in detail. In this process, two membrane-enclosed organelles recognize and adhere to one another, then their boundary membranes fuse to form a single organelle.
1. Controlling the progress of membrane fusion
This has yielded insights about how the fusion complex is assembled and how it triggers fusion (Schwartz and Merz, 2009). We obtained strong evidence that the fusion complex assembles through a two-stage “zippering” mechanism, and we found that defective complexes can be rescued by an accessory protein. Although done with yeast lysosomes, these experiments highlight general principles that will likely hold for other fusion events such as neurotransmission.
2. Mechanisms of membrane tethering
While tremendous effort has been focused on the biochemical reconstitution of fusion using purified components, almost no similar work has been done on tethering reactions. We therefore developed a reconstitution system to study tethering steps regulated by small signaling proteins called Rabs. It was previously thought that Rabs always act in concert with additional proteins to drive tethering. Instead, we discovered that at least some Rabs are functionally autonomous, and can tether membranes all by themselves. We are also using these methods to study more elaborate tethering mechanisms.
3. Global control of endocytic traffic by Vps-C complexes
These amazing protein complexes appear to act with Rab proteins as master regulators that organize all membrane traffic through late endosomal and lysosomal organelles, and in so doing control the size and number of these organelles. Vps-C complexes interact with Rab proteins in tethering, with the core fusion machinery, and with protein modification systems. We elucidated how the different domains within these complexes are arranged, we identified several sites where Vps-C complexes and Rab proteins interact, and we are now working to understand the underlying logic of these complexes and how it is linked to membrane traffic. Over the longer term our goal is to understand how in differentiated cells these mechanisms are regulated, and how they are harnessed and modified to carry out specialized functions such as antigen sampling and presentation. We are also beginning to study the mechanisms through which endoslysosomal organelles interact with organelles such as the lipid droplets where fats and sterols are stored.
