The abundance of certain sub-cellular compartments is dramatically altered by changes in metabolic state; therefore it seems reasonable that organelle biogenesis in general would be responsive to changes in cell physiology. The constitutive secretory pathway, essential for the synthesis and delivery of proteins and lipids to the cell surface as well as for the biogenesis of the organelles of the endo-membrane system, is a highly dynamic network of membrane-bound compartments and thus is a prime target for physiological regulation. While extensive research has yielded a detailed molecular description of a number of individual membrane trafficking steps, research aimed at understanding how these pathways are regulated and integrated is just beginning. The larger goal of our resesarch is to elucidate the regulatory mechanisms that allow the secretory pathway to respond to changes in cell physiology. At one end of the spectrum, we hope to identify the intracellular signals, presumably components of signal transduction pathways, that mediate alterations in membrane trafficking. At the other end of the spectrum, we hope to determine how the signals, generated in response to physiological stimuli, impinge on transport. This will consist of identifying the transport factors whose activities are modulated by changing phsyiological conditions as well as investigating the mechanism by which their activities are modulated.

To identify potential regulators of membrane trafficking, we are taking two broadly complementary approaches. In the first, we are using a cell-free morphological assay that reconstitutes several steps comprising the early secretory pathway. Such an assay allows a biochemical dissection of the factors that mediate specific sub-reactions underlying transport through the pathway. For instance, vesicle formation from the endoplasmic reticulum (ER), mediated by the cytoplasmic coat protein complex (COPII) binding to cargo molecules at ER exit sites, can be monitored at the light level by visualizing the assembly of COPII subunits at exit sites. Likewise, changes in ER membrane morphology are monitored by visualizing the distribution of resident ER membrane proteins. Using such assays, we have biochemically purified and identified NDPKB, an isoform of mammalian Nucleoside Diphosphate Kinase, as a contributing factor in both ER membrane extension and COPII-mediated ER export.

The second approach to identifying novel regulators involves a large-scale screen to identify proteins required for the integrity and function of organelles of the early secretory pathway.  Short interfering (si) RNAs that target the degradation of specific residents of the ER and ER-to-Golgi intermediate compartment (ERGIC) are introduced into living cells and their effects monitored using a variety of morphological assays.  Through such a knockdown screen, we have begun to identify several proteins required, not only for the function of the early secretory pathway, but also for the maintenance of ER and ERGIC structure. The precise mechanism by which these newly identified players functions to maintain both the structure and function of the ER and other membrane compartments will be elucidated using both live cell and in vitro systems. In the first, knockdown and gene replacement will be used to identify regions of the identified protein that are critical for each defined function. In the second, the molecular mechanism by which the critical identified regions function will be explored using a variety of biochemical assays. Through these combined complementary approaches, we hope to define the regulatory pathways that mediate regulated trafficking through the early secretory pathway.

Morin-Leisk J and Lee TH. Nucleotide dependent self-assembly of Nucleoside Diphosphate Kinase (NDPK) in vitro. Biochimica et Biophysica Acta, 2008; In press.

Baughman C, Morin-Leisk J, Lee TH. Nucleoside Diphosphate Kinase B (NDPKB) scaffolds endoplasmic reticulum membranes in vitro. Exp Cell Res 2008; In press.

Kapetanovich L, Baughman C, Lee TH. Nm23H2 Facilitates Coat Protein Complex II Assembly and Endoplasmic Reticulum Export in Mammalian Cells. Molecular Biology of the Cell 2005;16(2):835-48.

Bachert C, Lee TH, Linstedt AD. Lumenal endosomal and Golgi-retrieval determinants involved in pH-sensitive targeting of an early Golgi protein. Mol Biol Cell 2001; 12(10)3152-60.

Lee TH, Linstedt AD. Potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-Golgi transport revealed by protein kinase inhibitor H89. Molecular Biolology of the Cell 2000; 11(8):2577-90.

Lee TH, Linstedt AD. Osmotically induced cell volume changes alter anterograde and retrograde transport, Golgi structure, and COPI dissociation. Molecular Biology of the Cell 1999; 10(5):1445-62.