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Davis NgAssociate Professor of Biochemistry and Molecular Biology 408 South Frear Laboratory, University Park, PA 16802 A.B. in molecular biology, University of California-Berkeley Ng Lab Web Site |
Biogenesis and regulation of the early secretory pathway
A fundamental problem of cell biology is the biogenesis and maintenance of intracellular organelles. Eukaroytic cells are organized by segregating essential cellular functions into discrete compartments composed of proteins and lipids. Consequently, establishing and maintaining order requires: 1) mechanisms to synthesize and localize molecules tospecific destinations and 2) mechanisms to monitor and regulate the state of individual organelles. My research interests center on these two vital aspects of cellular function and how they relate to the endoplasmic reticulum (ER). The ER is the gateway to the secretory pathway and the site of biosynthesis for all secreted and resident proteins along the route. Proteins that utilize as well as constitute the secretory pathway are synthesized from cytoplasmic ribosomes and targeted for translocation across ER membranes by specific signal sequences. In the ER lumen, polypeptides are processed functional proteins by any of several maturation steps including N-linked glycosylation, disulfide bond formation, folding and oligomeric assembly prior to transport to its final destination. My laboratory focuses on these early steps with the goal to understand the mechanisms that mediate and regulate the biosynthesis of secretory pathway proteins. The model organism is the budding yeast Saccharomyces cerevisiae. It is ideal for these studies since it offers powerful genetic approaches and is amenable to biochemical analysis. In addition, it is currently the only eukaryotic organism with a known genomic sequence.
Dissection of multiple protein translocationpathways to the ER.
Targeting of secretory proteins in S. Cerevisiae was thought to be distinct mechanistically from higher eukaryotes because genetic and biochemical approaches had failed to identify a mammalian-like signal recognition particle (SRP). This view changed when yeast SRP homologs were later identified and shown to beinvolved in protein translocation across the ER. Surprisingly, these components were shown to be non-essential and required for only a subset of proteins suggesting the possible existence of multiple pathways. Using a genetic approach, mutants for specific for SRP-independent translocation were isolated. These and SRP-specific mutants led to the genetic and biochemical dissection of two parallel translocation pathways. To address the significance of two pathways, a range of substrates were examined with each falling into the following categories: SRP-dependent, SRP-independent or requiring both pathways. Using this system, it was demonstrated that signal sequences are not universal in nature, but contain specific structural information used to sort substrates to their respective pathways. The characterization and dissection of parallel translocation pathways lay the groundwork for studies addressing how the overall flux of proteins enters the secretory pathway. This question will be approached from two perspectives: 1) What specific characteristics set apart SRP-dependent and SRP-independent signal sequences? 2) What are the structure-function relationships that allow recognition factors to bind productively to one class of signal sequence and not the other?
An ER to nucleus signal transduction pathway.
External stimuli (e.g., ligands, metabolites, antigens) are detected and processed in cells by signal transduction pathways that can lead to the regulation of specific genes. Within the cell, the state of the ER is monitored in analogous fashion by an intracellular signal transduction pathway. For example, agents causing the misfolding of proteins in the ER lumen lead to increased expression of specific genes (only a few of which have been identified) through a phenomenon termed the unfolded protein response pathway (UPR). Although the products of these genes help alleviate some negative effects of some chemically induced stress, the precise physiological function of the pathway remains unknown. Recently, a number of the genes required for the UPR signaling pathway was cloned. Interestingly, none of these genes are essential nor show any obvious phenotypes when deleted singly or in combination apart from the inability to induce expression of target genes. This might have been expected given that under normal conditions, target genes are not induced. To reveal the physiological role of the pathway and to identify genes involved in ER protein folding, a genetic screen was devised to isolate mutants that are dependent on the UPR pathway for viability. Because the pathway is not activated under normal conditions, mutations that render it essential reflect defects in functions that are modulated by the pathway. To date, the recessive mutants fall into 16 complementation groups with the screen not yet saturated. These mutants immediately become a valuable resource for a variety of reasons. First, each disrupts a cellular function leading to the activation of the pathway. Since it is not known how the pathway is triggered, analysis of the mutants will provide insight into the mechanism of UPR activation. Second, as UPR pathway-regulated genes are among the expected targets, it is likely that many, if not all, will be identified by the screen. This notion is underscored by the identity of a novel UPR target gene required for glycosylation among the group. Third, since it is known that misfolded proteins in the ER can activate the pathway many of the mutants will be defective in protein folding. Thus, the products of the screen will be instrumental in identifying factors that influence protein folding and in turn provide unique tools to study ER protein maturation. Indeed, our analysis of the panel of mutants has revealed functions in many facets of protein maturation. The next objective is to identify all the mutant genes. This work will reap immediate benefits because some mutations will map to known genes and thus provide important clues at functions regulated by the UPR pathway. This route has been pursued by testing cloned candidate genes for complementation (e.g., UPR-regulated) or by directly cloning the genes. Strikingly, most of the genes cloned so far are novel and underscores our incomplete understanding these essential cellular functions. With these reagents in hand, our goal is to reveal the molecular mechanisms of secretory protein biogenesis and the role of the unfolded protein response in regulating these functions.
Representative Publications:
- Ng, D. T. W., E. D. Spear, and P. Walter. 2000. The unfolded protein response pathway regulates multiple aspects of secretory and membrane protein biogenesis and endoplamic reticulum quality control. J. Cell Biol. 150:77-88.
- Spear, E. D. and D. T. W. Ng. 2001. The unfolded protein response: no longer just a special teams player. Traffic. 2:515-523.
- Ng, D. T. W. 2001. Interorganellar signal transduction: The arrest of secretion response. Dev. Cell. 1:319-320.
- Vashist, S., W. Kim, W. Belden, E. D. Spear, C. Barlowe, and D. T. W. Ng. 2001. Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding. J. Cell Biol. 155:355-367.
- Helenius, J., D. T.W. Ng, C. L. Marolda, P. Walter, M. A. Valvano and M. Aebi. 2002. The RFT1 protein is required for the translocation of lipid-linked oligosaccharides across the membrane of the endoplasmic reticulum. Nature. 415:447-450.
- Vashist, S., C. G. Frank, C. A. Jakob, and D. T.W. Ng. 2002. Two Distinctly Localized P-Type ATPases Collaborate to Maintain Organelle Homeostasis Required for Glycoprotein Processing and Quality Control. Mol. Biol. Cell. 13:3955-3966.
- Spear, E. D. and D. T. W. Ng. 2003. Stress tolerance of misfolded carboxypeptidase Y requires maintenance of protein trafficking and degradation pathways. Mol. Biol. Cell. 14:2756-2767
- Vashist and D. T. W. Ng. 2003. Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. Submitted.