William Dowhan, Ph.D.
Professor and John S. Dunn Chair in Biochemistry & Molecular Biology


Description of Research

STRUCTURE, ASSEMBLY AND FUNCTION OF CELL MEMBRANE COMPONENTS

My laboratory for the past 40 years has carried out studies on the two major components of cell membranes–Phospholipids and Proteins. Early studies in Escherichia coli focused on purification and characterization of membrane-associated enzymes responsible for synthesis of the major phospholipid classes. Molecular genetic approaches are currently used to construct strains of E. coli and Saccharomyces cerevisiae in which membrane phospholipid composition can be regulated in a dose-dependent and temporal manner to define the role of specific phospholipids in cell function.

Through variation of phospholipid composition and expression of foreign lipids in E. coli, the following roles for phospholipids have been defined: structure, topological organization and function of membrane proteins; organization and function of the cell division and DNA replication machinery; organization and function of lipid domains in the membrane; export of proteins across the inner membrane; control of regulated membrane permeability. In yeast mitochondria important roles have been delineated for the phospholipid cardiolipin in organization and function of the respiratory chain.

E. coli phospholipid biosynthetic pathway (blue) plus synthesis of lipids by inserted foreign genes (red) Identified cloned genes indicated for each step. See Biochim. Biophys. Acta 1831: 471-494 (2013).

E. coli phospholipid biosynthetic pathway (blue) plus synthesis of lipids by inserted foreign genes (red) Identified cloned genes indicated for each step. See Biochim. Biophys. Acta 1831: 471-494 (2013)

S. cerevisiae synthesis of mitochondrial phospholipids. Cloned genes for each step shown. See chemphyslip.2013.10.012

S. cerevisiae synthesis of mitochondrial phospholipids. Cloned genes for each step shown. See chemphyslip.2013.10.012

Membrane Protein Topogenesis

A current important focus in Membrane Biology is to understand integral a-helical membrane protein topogenesis. How is the final orientation of protein transmembrane domains (TMDs) relative to the plane of the phospholipid orchestrated? Through the use of strains of E. coli in which the synthesis of the major phospholipid, phosphatidylethanolamine (PE), can be regulated at steady state and temporally during the cell cycle, we have uncovered a central role of membrane phospholipid composition as a determinant of membrane protein topological organization. First, using lactose permease (LacY, the paradigm of secondary transporters throughout nature) and other secondary transporters, we showed that in the absence of PE these transporters are mis-organized with respect to the orientation of their TMDs. Second, we showed that LacY topological organization could be changed post-assembly in response to changes in the PE content of membranes. Third, multiple topological forms of LacY co-exist at intermediate levels of PE. Fourth, although these forms are not in rapid equilibrium with each other, the relative levels of these forms can be changes in response to a change in membrane PE levels. Finally, the above in vivo observations have been reproduced in an in vitro proteoliposome system composed of only LacY and phospholipids. Thus, initial topological organization, existence of multiple topological conformers, and post-assembly topological re-organization of TMD orientation are thermodynamically driven processes dictated solely by direct lipid-protein interactions. Although protein molecular chaperones and other cellular components may be involved in vivo in these processes, they are not absolutely necessary. Therefore, in more complex eukaryotic cells changes in local lipid environment temporally (movement in and out of lipid rafts) or during intracellular vesicular trafficking can change the organization and function of a membrane protein. See the work of Mikhail Bogdanov and Heidi Vitrac for more details.

Topological organization of LacY as a function of membrane PE content. See J. Biol. Chem. 287: 37939-37948 (2012)

Topological organization of LacY as a function of membrane PE content. See J. Biol. Chem. 287: 37939-37948 (2012)

Selected References

Dowhan, W. and Bogdanov, M.: Lipid-dependent membrane protein topogenesis. Annu. Rev. Biochem. 78:515-40 (2009)

Dowhan, W.: A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function. Biochim. Biophys. Acta, 1831: 471-494 (2013).

Bogdanov, M. and Dowhan, W.: Lipid-dependent generation of a dual topology for a membrane protein. J. Biol. Chem. 287: 37939-37948 (2012)

Vitrac, H., Bogdanov, M. and Dowhan, W. In vitro reconstitution of lipid-dependent dual topology and post-assembly topological switching of a membrane protein. Proc. Nat’l. Acad. Sci., U.S.A 110: 9332-9337 (2013)

Bogdanov, M., Dowhan, W. and Vitrac, H.: Lipids and topological rules governing membrane protein assembly. Biochim. Biophys. Acta, in press (2014)

Role of Cardiolipin in Mitochondrial Respiration

Yeast mutants (∆pgs1) lacking both phosphatidylglycerol (PG) and cardiolipin (CL) are severely compromised in structural organization of mitochondria and completely lack respiratory function, Therefore, such mutants only grow by fermentation. However, mutants (∆crd1) lacking of only CL with increased levels of the CL precursor PG are only compromised for growth on non-fermentable carbon sources. Upon further investigation of ∆crd1 mutants, we determined that the organization of the respiratory chain into supercomplexes of the individual respiratory Complexes III (cytochrome bc1) and IV (cytochrome c oxidase) was the molecular basis for the poor growth on non-fermentable carbon sources. We determined by electron cryo-microscopy the first 3-dimensional density map the of the III2-IV2 supercomplex. Docking of the crystal structures of Complexes III and IV onto the density map generated a pseudo-atomic model of the supercomplex. This model showed the orientation of the individual complexes with respect to each other, gaps at the transmembrane domain interface of the complexes that lie within the lipid bilayer, and the distance between the cytochrome c binding sites of Complex III and Complex IV. The gaps are consistent with the approximately 50 molecules of CL associated with the supercomplex. This number is in excess over the CL molecules known to be integral to the individual complexes. The distance between the cytochrome c binding sites is consistent with the observed CL-dependent channeling of this substrate Complexes III and IV as we previously demonstrated. Finally, reconstitution of the supercomplex from individual purified Complexes III and IV was achieved wholly dependent on addition of CL over that integral to the individual complexes. These results establish a specific role for CL in organization of a function respiratory chain and supports a similar role for CL in mammalian mitochondria where reduced levels of CL in Barth Syndrome displays as serious defects in respiratory function. For structural details see work of Eugenia Mileykovskaya.

Selected References

Zhang, M, Mileykovskaya, E. and Dowhan, W.: Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria. J. Biol. Chem. 280: 29403-29408, 2005

Mileykovskaya, E., Penczek, P.A., Fang, J., Mallampalli, V. K. P. S., Sparagna, G. C.and Dowhan, W. Arrangement of the Respiratory Chain Complexes in Saccharomyces cerevisiae Supercomplex III2IV2 Revealed by Single Particle Cryo-Electron Microscopy (EM). J. Biol. Chem. 287: 23095-23103 (2012)

Bazán, S, Mileykovskaya, E., Mallampalli, V. K. P. S., Heacock, P. N, Sparagna, G.C.and Dowhan, W. Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV. J. Biol. Chem. 288: 401-411 (2013)

Mileykovskaya, E. and Dowhan, W.: Cardiolipin-dependent formation of mitochondrial respiratory supercomplexes. Chem. Phys. Lipids in press (2013)

Contact Information

William.Dowhan@uth.tmc.edu

UTHealth Medical School
Department of Biochemistry and Molecular Biology
6431 Fannin Street, MSB 6.224
Houston, Texas 77030

713-500-6051 Direct  713-500-0652 Fax

Education

A.B. Chemistry 1964 - Princeton University, Princeton, NJ

Ph.D. Biochemistry 1969 - University of Californian, Berkeley, CA

Postdoctoral Fellow, Am Cancer Soc 1969-72 - Harvard Medical School, Boston, MA

Research Interests

Structure, Assembly and Function of Cell Membranes, Lipids as Determinants of Membrane Protein Structure and Function, Role of Cardiolipin in Mitochondrial Organization and Function

Publications

A retrospective: use of Escherichia coli as a vehicle to study phospholipid synthesis and function

Dowhan W.

Biochim Biophys Acta. 1831:471-94 (2013)

PMID: 22925633

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Cardiolipin-dependent formation of mitochondrial respiratory supercomplexes

Mileykovskaya E, Dowhan W.

Chem Phys Lipids. 2013. doi:pii: S0009-3084(13)00150-3. 10.1016/j.chemphyslip.2013.10.012.

PMID: 24220496

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Lipids and Topological Rules Governing Membrane Protein Assembly

Mikhail Bogdanov, William Dowhan and Heidi Vitrac

Biochim. Biophys. Acta 2013 Dec 13. pii: S0167-4889(13)00426-6. doi: 10.1016/j.bbamcr.2013.12.007. [Epub ahead of print]

PMID: 24341994

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