Research Projects

My research program is focused on understanding protein and lipid transport through the early secretory pathway in eukaryotic cells. This is an essential process that initiates the delivery of proteins to intracellular organelles and to the cell surface for secretion. Because of the advantage for genetic analyses, we investigate this process mainly in the yeast Saccharomyces cerevisiae. Our primary interest is elucidating the molecular mechanisms that underlie vesicular transport between the endoplasmic reticulum (ER) and Golgi complex. Transport between these compartments is mediated by membrane vesicles, termed COPII vesicles, that bud from the ER and fuse with and/or form the Golgi complex.

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Our studies combine molecular genetics, proteomics and microscopy with cell free assays that measure COPII-dependent transport. This cell free transport reaction proceeds through the biochemically distinct stages of COPII-dependent vesicle budding, Uso1p-dependent vesicle tethering and SNARE protein-dependent membrane fusion. We have reproduced these stages with isolated membranes and purified soluble molecules. A long-term goal of my research program is to reconstitute distinct sub-reactions in ER/Golgi transport with defined protein and lipid fractions for elucidation of catalytic mechanisms. The ongoing research projects in my lab are focused on the vesicle budding, Uso1p-dependent vesicle tethering and membrane fusion stages described below.

COPII-Dependent Vesicle Budding.
The coat protein complex II (COPII) catalyzes transport vesicle formation from the ER and segregates secretory proteins from ER-resident proteins. To identify additional components of the ER/Golgi transport machinery, we scaled up our in vitro budding assay and obtained sufficient quantities of COPII vesicles for identification of abundant vesicle proteins. This approach uncovered a novel set of integral membrane ER vesicle proteins (Erv proteins) that included Erv14p, Erv24p/Emp24p, Erv25p, Erv26p, Erv29p, Erv41p and Erv46p.

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Although these proteins are conserved in nature, little functional information had been reported and their amino acid sequences provided no insight into activity. We have used molecular genetic approaches combined with our in vitro transport assays to determine functional roles for these proteins. Our results indicate that the Erv proteins are dynamically localized to early compartments of the secretory pathway and function in different aspects of intracellular transport. In one informative set of experiments, we demonstrated that Erv29p acts in collecting soluble secretory proteins into COPII vesicles. Such a receptor-like activity had been hypothesized but not molecularly defined. Based on our studies, we have proposed that Erv29p and some of the other Erv proteins link specific secretory cargo to the COPII vesicle coat. We continue to study the mechanisms of Erv protein function in transport between the ER and Golgi compartments.

Uso1p-Dependent Vesicle Tethering.
Using our cell free vesicle fusion assay, we isolated a set of soluble factors (Uso1p, Sec18p and LMA1) that replaced the requirement for cytosol in fusion of isolated COPII transport vesicles with Golgi membranes. Further experimentation indicated that Uso1p acted early in this fusion reaction and produced a "tethered" intermediate that could be chased to fusion upon addition of the other soluble factors. Having distinct vesicle tethering and fusion assays allowed us to determine the stage at which other genetically defined membrane bound proteins operate using thermosensitive mutations and/or specific neutralizing antibodies to block activity.

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We found that the Rab GTPase Ypt1p is required for vesicle tethering whereas requirements for ER/Golgi SNARE proteins in tethering were not detected. Moreover, our data show that Ypt1p activity is asymmetrically provided by the acceptor Golgi membranes and not the COPII vesicles, as previously thought. More recently we have found that membrane bound Ypt1p is required for Uso1p membrane association and have detected a solubilized complex containing. Uso1p and Ypt1p. We hypothesize that Uso1p tethers vesicles by binding to factors on the surface of COPII vesicles and to a Ypt1p protein complex on acceptor membranes. We continue to investigate components on vesicles and Golgi membranes that are required for vesicle tethering.

SNARE-Dependent Membrane Fusion.
Fusion of tethered COPII vesicles depends on a set of membrane bound SNARE proteins (Sed5p, Sec22p, Bet1p and Bos1p) plus Sly1p, Got1p and probably additional uncharacterized factors. Cognate sets of SNARE proteins are known to from stable complexes through assembly of their SNARE motifs into a parallel four-helix coiled-coil structure. Assembly of SNARE complexes in trans, i.e. from donor and acceptor membranes, somehow mediates fusion of intracellular membranes. We have detected the ER/Golgi SNAREs and SNARE associated proteins on both COPII vesicles and acceptor membranes.

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However, their functional requirements are asymmetric in keeping with what one might expect for a heterotypic fusion event. We have also found that under certain conditions, Sec22p is dispensable for growth because a related SNARE protein that operates in later stages of the secretory pathway, Ykt6p, can functionally substitute for Sec22p. These and other findings indicate a degree of flexibility in the SNARE protein requirements for ER/Golgi transport and suggest that combinatorial mechanisms using both upstream-tethering elements and SNARE proteins are needed for efficient membrane fusion. We continue to explore the hypothesis that a relay between the tethering and fusion machinery, possibly governed by Ypt1p, is required for specificity in vesicle fusion. In vitro assays to monitor assembly of ER-Golgi SNARE complexes during membrane fusion are also under development.