Robert C. Piper, Ph.D.

Associate Professor

Washington University, St. Louis, 1992

Research Interests

Ubiquitin Mediated Trafficking to the Lysosomal Degradative Pathway
We are interested in the sorting events that direct cell surface receptors and other membrane proteins for degradation in lysosomes. After internalization from the plasma membrane, these membrane proteins are incorporated into vesicles that bud into the lumen of the endosome to create a multivesicular body or multi-vesicular endosome. Once these lumenal membranes are delivered to lysosomes, both the cytosolic and lumenal domains of receptor proteins are destroyed by the concerted action of lipases and proteases. The proper exclusion or inclusion of proteins into this degradative sorting pathway is a crucial process within the endosomal system. We have shown that ubiquitination of membrane proteins drives their sorting into the lumenal vesicles of the endosome. This provides an elegantly simple mechanism for acutely and selectively sorting proteins for degradation in lysosomes. We have sought to understand how ubiquitin serves as a specific sorting signal by mapping the sorting motifs present on ubiquitin and identifying and characterizing "ubiquitin-sorting receptors". We have identified a protein complex composed of Vps27 and Hse1, which sorts ubiquitinated cargo proteins into multivesiculated bodies. We have also found the clathrin-associated GGA coat proteins act as ubiquitin-sorting receptors, directing ubiquitinated cargo from the TGN to endosomes prior to its incorporation into multivesiculated bodies. We now want to identify and understand how other proteins participate in ubiquitin-dependent sorting events and to evaluate how this process may be regulated by global physiological events such as hormonal stimulation or nutritional status. We also want to understand how these receptors coordinate their activity with other proteins responsible for the formation of the lumenal vesicles themselves. Our general approach has been to use the yeast model system where we can combine searches for particular interacting partners important for ubiquitin-dependent sorting with structural studies to help ascertain the function of these interactions in vivo.

Tethering factors in the coordinated control of endosome movement and fusion.
Transport of proteins to lysosomes also relies on the movement and fusion of early endosomes, late endosomes and lysosomes. While some of the protein machinery that controls these events has been described, we have yet to understand how these proteins actually function together to coordinate endosome movement and positioning with distinct membrane fusion events. Another focus of our lab is to find out how endosomal fusion is controlled and how it might be coordinated with cytoskeletal functions. Our earlier work set out to define which SNARE proteins catalyzed discrete fusion steps in the endocytic pathway sine SNARE proteins are central to the fusion process. We then focused on understanding how this core fusion machinery might be regulated by identifying a set of "tethering" factors that catalyze endosomal fusion. These proteins, collectively designated as the mHOPS complex, are orthologues of yeast proteins required for HOmotypic fusion of vacuoles and vacuole Proteins Sorting. Unexpectedly, we found the mHOPS proteins work at numerous fusion steps in the endocytic pathway and that different subunits of this complex associate with the actin or the microtubulule cytoskeleton. These data suggested to us that the mHOPS tethering complex integrates cytoskeletal/organelle interaction with SNARE mediated fusion events ^9,10. Recently, we found this complex directly associates with organelle-associated myosin motors. Our present goal is to determine not only how the mHOPS complex is assembled and how it interacts with SNARE complexes and GTPases but also what aspect of endosomal biogenesis is provided by the interaction of mHOPS with the cytoskeleton.

Representative References

1. Bilodeau, P.S. et al. (2002) The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting. Nature Cell Biol 4 (7), 534-539

2. Bilodeau, P.S. et al. (2003) Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome. J Cell Biol 163 (2), 237-243

3. Scott, P.M. et al. (2004) GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network. Nature Cell Biol 6 (3), 252-259

4. Richardson, S.C. et al. (2004) Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Mol Biol Cell 15 (3), 1197-1210