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Cell Signaling and Membrane Trafficking
Summary: Scott Emr's lab studies the regulation of cell signaling and membrane trafficking pathways by phosphoinositide kinases, protein kinases, selective ubiquitin modifications, and vesicle-mediated transport reactions.
All eukaryotic cells maintain an elaborate system of vesicular transport pathways that convey cargo in and out of the cell via the endocytic and secretory systems. Our long-term goal has been to define the complex regulatory processes that ensure the temporal and spatial specificity of these membrane trafficking systems. This past year we have focused on two major research interests: (1) endocytic trafficking and receptor down-regulation and (2) phosphoinositide lipid- and ubiquitin-dependent membrane trafficking pathways.
Endocytosis and Receptor Down-Regulation
The endocytic pathway is required for many key cellular processes, including down-regulation of activated cell surface receptors and the sorting of plasma membrane proteins to the lysosome. A subset of late endosomes contain internal vesicles and are referred to as multivesicular bodies (MVBs). MVB vesicles form by a membrane invagination process in which the vesicles bud into the lumen of the endosome. Our work has provided insights into the mechanism and regulation of this essential transport pathway. Entry of cargo into the MVB pathway, a highly regulated event, requires both cis- and trans-acting factors. Ubiquitination of endosomal membrane proteins serves as a positive sorting signal for inclusion in the intralumenal MVB vesicle. We have cloned and characterized more than a dozen transport components in the MVB-sorting pathway. Several of these proteins assemble into a set of protein-sorting complexes referred to as the ESCRT complexes (endosomal sorting complexes required for transport), the sequential activities of which are required for the recognition and sorting of ubiquitin-modified MVB cargo proteins. Therefore, an obvious mechanism by which to control entry into the MVB pathway is at the level of ubiquitination. Understanding the machinery responsible for the ubiquitination of MVB cargoes is of critical importance for understanding how the MVB pathway is regulated and how MVB cargo is selected.
During the past year, we have identified new yeast mutants that missort MVB cargoes. One of these contains a point mutation in the ubiquitin ligase Rsp5. The mutant exhibits a defect in the ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS). RSP5 encodes a HECT domain–containing ligase that functions as a ubiquitin ligase at multiple locations within the cell, including the plasma membrane, endoplasmic reticulum, Golgi, endosome, and mitochondria. Therefore, Rsp5 must be selectively recruited to/activated at diverse membrane compartments within the cell. This recruitment/activation of Rsp5 is likely to be highly regulated and dependent on both protein-protein and protein-lipid interactions. Indeed, Rsp5 contains motifs responsible for driving interactions with both proteins and lipids. In addition to the carboxyl-terminal HECT domain, the amino-terminal portion of Rsp5 contains a C2 domain that binds charged lipids and three WW protein–interaction domains capable of binding proline-rich sequences. We found that both the WW2 and WW3 domains of Rsp5 are required for the ubiquitination and sorting of pCPS into the MVB pathway, suggesting a conserved role for these domains in the ubiquitination of MVB cargoes.
Besides localizing to the appropriate membrane compartment, Rsp5 must selectively recognize appropriate target proteins for ubiquitination. This is clearly demonstrated at the endosome, where the MVB cargoes such as pCPS are efficiently ubiquitinated, whereas others, which contain multiple lysines in its cytoplasmic tail, fail to receive this modification (and do not become MVB cargoes). It would, therefore, seem that the activity of Rsp5 is dictated only in part by its subcellular localization. There may also be a conserved motif within Rsp5 substrates that is directly recognized by Rsp5. Thus far, we have screened a small number of mutants in our genetic study. Extending this genetic screen should help to identify other gene products required for MVB sorting, as well as additional genes that regulate Rsp5 localization and cargo recognition.
The recognition of ubiquitinated cargoes (receptor proteins) in the MVB pathway requires the function of three distinct protein complexes: ESCRT-I, -II, and -III. ESCRT-I recruits the downstream ESCRT-II and ESCRT-III complexes. After the ESCRTs have been recruited to the endosome membrane, the AAA-type ATPase Vps4 binds ESCRT-III and, following MVB vesicle formation, catalyzes the dissociation of the ESCRT protein complexes in an ATP-dependent manner for further rounds of protein sorting. We recently determined the crystal structure for the core of the yeast ESCRT-II complex. The three-lobed structure contains one molecule of the Vps protein Vps22, the carboxyl-terminal domain of Vps36, and two molecules of Vps25. The amino-terminal coiled coil of Vps22 and the flexible linker leading to the ubiquitin-binding NZF domain of Vps36 both protrude from the tip of one lobe of the complex. The structure suggests how ubiquitinated cargo could be passed between ESCRT components of the MVB pathway through the sequential transfer of ubiquitinated cargo from one complex to the next.
Our working model suggests that larger oligomers of ESCRT complexes could form a highly organized scaffold. Such a scaffold could prevent the ubiquitinated cargo from diffusing away from one of the low-affinity binding domains in the complex. A mechanistic understanding of this pathway will require additional structures of the remaining complexes as well as those of the higher order complexes of complexes. The resolution of the first of these structures provides a glimpse into the mechanism of ESCRT complex formation and a foundation for understanding cargo sorting and MVB vesicle formation.
Of particular medical relevance, the ESCRT machinery has been shown to play an essential role in HIV viral budding. Topologically, the formation of MVB vesicles at the endosome and viral budding at the plasma membrane are similar. Wesley Sundquist (University of Utah) and colleagues demonstrated that depletion of ESCRT-I by siRNA (small interfering RNA) causes defects in the budding of viral particles. These observations suggest a model in which HIV hijacks the ESCRT machinery to drive the formation of viral particles. The ESCRT machinery therefore represents a new set of candidate targets for the development of antiviral drugs.
Phosphoinositide Signaling and Membrane Trafficking
We previously discovered that phosphoinositide (PI) lipids play an essential role in the regulation of membrane trafficking in both yeast and mammalian cells. PIs regulate diverse cellular processes, including cell growth, survival, differentiation, cytoskeletal organization, and membrane trafficking. Our long-term goal has been to understand how synthesis and turnover of PIPs (PI phosphates) are temporally and spatially regulated by lipid kinases and phosphatases. In addition, we have been using genetic and biochemical techniques to identify downstream targets/effectors for the structurally distinct PIPs.
To extend our understanding of the PI kinase signaling networks in yeast, we initiated a collaboration with Charles Boone's laboratory (University of Toronto) to employ synthetic genetic array (SGA) analysis to identify new genes acting in PI kinase signaling pathways. We have applied this approach to combine PI kinase temperature-sensitive (ts) mutants generated in the lab with an array of ~4,700 unique yeast deletion mutants. Initial experiments have uncovered numerous synthetic lethal interactions. Many of these genetic interactions have been verified by direct genetic analysis. Of particular interest, one of the PI 4-kinases in yeast that functions in the secretory pathway exhibited genetic interactions with several genes required for secretory function, providing insights into candidate downstream effectors of PI4P.
In another SGA screen, we uncovered numerous new candidate effectors and regulators of PI4,5P2 in yeast. We identified Slm1, a previously uncharacterized PI4,5P2-binding protein. Slm1 and its homolog Slm2 are required for actin cytoskeleton polarization and viability. Coimmunoprecipitation experiments revealed that Slm1 interacts with a component of TORC2, a Tor2 kinase-containing complex, which also regulates the actin cytoskeleton. Consistent with these findings, phosphorylation of Slm1 and Slm2 depends on TORC2 protein kinase activity, both in vivo and in vitro, and Slm1 localization requires both PI4,5P2 and functional TORC2. Our data suggest that Slm1 and Slm2 function downstream of PI4,5P2 and the TORC2 kinase pathway to control actin cytoskeleton organization. Further study of the genetic interactions found using the SGA approach should reveal novel components in each of the PI kinase signaling pathways.
The Saccharomyces cerevisiae synaptojanin-like (Sjl) proteins function as PI phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast Sjl proteins remains unclear. During this past year, we have shown that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, we found that the SH3 domain in Abp1 physically associates with Sjl2 through its proline-rich motif. Abp1 therefore appears to act as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Our studies suggest that PI metabolism by the Sjl proteins coordinately directs actin dynamics and membrane invagination during endocytosis.
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