Tom Stevens

Professor of Chemistry

B.A., San Francisco State University;
M.S., San Francisco State University;
Ph.D., California Institute of Technology

Member of:
• Institute of Molecular Biology

Research Interests

Stevens’ research group is concerned with the process of protein sorting and membrane assembly in yeast cells. Using yeast molecular genetics, we have identified a large number of genes required for the correct targeting and transport of proteins to the membrane-bounded organelle called the vacuole. These vacuolar protein sorting (VPS) genes have been found to encode proteins such as a dynamin-like GTPase, a protein-sorting receptor, a protein kinase, a lipid kinase, a RAS inhibitor-like protein, and an increasingly large number of proteins involved in transport vesicle targeting/fusion such as Rab-like GTPases and SNARE proteins. To characterize the function of some of these proteins we use biochemical, cell biological and molecular genetic approaches. Biochemical approaches are being used to isolate a number of the VPS proteins and to study the membrane-associated protein complexes in which they are found. We use mass spectrometry to identify the unknown proteins present in these sorting complexes.

The group also has a long-standing interest in the assembly, targeting, structure and function of the vacuolar H+-translocating ATPase (V-ATPase; see figure). The V-ATPase complex consists of fourteen subunits, and all but one of these are encoded by a single yeast gene. The large hydrophobic “a” subunit has two isoforms, Vph1 and Stv1, with the Vph1-associated V-ATPase complex localizing to the vacuole membrane and the Stv1-associated V-ATPase restricted to Golgi and endosomal membranes. The mechanism of differential localization of these two forms of the yeast V-ATPase is under active investigation in the lab. We are investigating the proteins responsible for maintaining this differential localization, as well as the protein-based signals that specify the distinct localizations.

We have also identified four genes that encode proteins required for V-ATPase complex assembly but are not themselves part of the final V-ATPase enzyme complex. These four proteins reside in the yeast cell endoplasmic reticulum and constitute the dedicated assembly machinery for the V-ATPase. A number of molecular genetic and biochemical approaches are being taken to characterize the assembly complex and to study the interaction of this assembly complex with V-ATPase subunits along the assembly pathway within the endoplasmic reticulum.

Figure 1 Schematic drawing of the yeast endomembrane pathways. Proteins exiting the Golgi complex can be transported by either the CPY pathway (arrow 1) through the prevacuole to the vacuole, or by the ALP pathway (arrow 2). Proteins can also reach the prevacuole by endocytosis (arrow 3), and proteins in the prevacuole transit a common pathway to the vacuole (arrow 5). Golgi membrane proteins such as DPAP A are retrieved from the prevacuole back to the Golgi (arrow 4).

The group also has a long-standing interest in the assembly, targeting, structure and function of the vacuolar H +-translocating ATPase (V-ATPase; see figure). The V-ATPase complex consists of fourteen subunits, and all but one of these are encoded by a single yeast gene. The large hydrophobic “a” subunit has two isoforms, Vph1 and Stv1, with the Vph1-associated V-ATPase complex localizing to the vacuole membrane and the Stv1-associated V-ATPase restricted to Golgi and endosomal membranes. These studies reveal that the targeting information for V-ATPase localization is found in the “a” subunit. The mechanism of differential localization of these two forms of the yeast V-ATPase is under active investigation in the lab. The group is investigating the proteins responsible for maintaining this differential localization, as well as the protein-based signals that specify the distinct localizations.

These researchers have also identified four genes that encode proteins required for V-ATPase complex assembly but are not themselves part of the final V-ATPase enzyme complex. These four proteins reside in the yeast cell endoplasmic reticulum and constitute a dedicated assembly machinery for the V-ATPase. A number of molecular genetic and biochemical approaches are being taken to characterize the assembly complex and to study the interaction of this assembly complex with V-ATPase subunits along the assembly pathway within the endoplasmic reticulum.

Figure 3 Assembly of the V 0 sector in the ER. Vma21p interacts with the proteolipid ring V-ATPase subunits of Vma3p (c), Vma11p (c’), and Vma16p (c’’). Vma12p and Vma22p form a complex on the ER membrane and interact with newly synthesized “a” subunit (Vph1p and Stv1p), and then Vma21p “escorts” this V 0 sector into vesicles budding from the ER membrane. Vma21p accompanies the V-ATPase to the Golgi complex, where its C-terminal KKXX ER retention signal is recognized by the COPI machinery and this assembly/escort factor is recycled to the ER. The assembled V-ATPase is then transported on to the endosome and/or vacuole.

Selected Publications

Complete List of Publications

Davis-Kaplan, S.R., Compton, M.A., Flannery, A.R., Ward, D.M., Kaplan, J., Stevens, T.H. and L.A. Graham (2006) PKR1 encodes an assembly factor for the yeast V-type ATPase. J Biol Chem. 281 (42): 32025-32035.

Compton, M.A., Graham, L.A. and T.H. Stevens (2006) Vma9p (subunit e) is an integral membrane V0 subunit of the yeast V-ATPase. J Biol Chem. 281(22):15312-9

Lottridge, J.M., Flannery, A.R., Vincelli, J.L. and T.H. Stevens (2006) Vta1p and Vps46p regulate the membrane association and ATPase activity of Vps4p at the yeast multivesicular body. Proc Natl Acad Sci U S A. 2006 Apr 18;103(16):6202-7

Bowers, K. and T.H. Stevens (2005) Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae.  Biochim. Biophys. Acta, 1744, 438-454.

Bowman E.J., L.A. Graham, T.H. Stevens, and B.J. Bowman (2004) The bafilomycin/concanamycin binding site in subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae. J Biol Chem 279:33131-8.

Malkus P., L.A. Graham, T.H. Stevens, and R. Schekman (2004) Role of vma21p in assembly and transport of the yeast vacuolar ATPase. Mol Biol Cell 15:5075-91.

Flannery A.R., L.A. Graham, and T.H. Stevens (2004) Topological characterization of the c, c', and c" subunits of the vacuolar ATPase from the yeast Saccharomyces cerevisiae. J Biol Chem 279:39856-62.

Graham L.A., A.R. Flannery, and T.H. Stevens (2003) Structure and assembly of the yeast V-ATPase. J Bioenerg Biomembr 35:301-12.

Kweon Y., A. Rothe, E. Conibear, and T.H. Stevens (2003) Ykt6p Is a Multifunctional Yeast R-SNARE That Is Required for Multiple Membrane Transport Pathways to the Vacuole. Mol Biol Cell 14:1868-81.

Conibear E., J.N. Cleck, and T.H. Stevens (2003) Links Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p. Mol Biol Cell 14:1610-23.

Conibear E. and T.H. Stevens (2002) Studying yeast vacuoles. Methods Enzymol. 351:408-32.

Kawasaki-Nishi S., K. Bowers, T. Nishi, M. Forgac, and T.H. Stevens (2001) The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis. J Biol Chem 276(50):47411-20.

Sagermann, M., T.H. Stevens, and B.W. Matthews (2001) Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae. PNAS 98(13):7134-9.

Gerrard, S.R., A.B. Mecklem, and T.H. Stevens (2000) The yeast endosomal t-SNARE, Pep12p, functions in the absence of its transmembrane domain. Traffic 1(1):45-55.

Conibear E. and T..H. Stevens (2000) Vps52p, Vps53p and Vps54p forms a novel multisubunit complex required for protein sorting at the yeast late Golgi. Mol Biol Cell 11(1):305-23.

Graham, L.A., B. Powell, and T.H. Stevens (2000) Composition and assembly of the yeast vacuolar H+-ATPase complex. J Exp Biol 203:61-70.

Gerrard, S.R., N.J. Bryant, and T.H. Stevens (2000) Vps21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment. Mol Biol Cell 11(2):613-26.

Gerrard, S.R., B.P. Levi, and T.H. Stevens (2000) Pep12p is a multifunctional yeast syntaxin that controls entry of biosynthetic endocytic and retrograde traffic into the prevacuolar compartment. Traffic 1(3):259-69.

Powell, B., L.A. Graham, and T.H. Stevens (2000) Molecular characterization of the yeast vacuolar H+-ATPase proton pore. J Biol Chem. 275(31):23654-60.

Bowers, K., B.P. Levi, F.I. Patel, and T.H. Stevens (2000) The sodium/proton exchanger Nhx1p is required for endosomal protein trafficking in the yeast Saccharomyces cerevisiae. Mol Biol Cell 11(12):4277-94.