Jurgen Heymann

Three-dimensional structure of a bacterial oxalate transporter.

The major facilitator superfamily (MFS) represents one of the largest classes of evolutionarily related membrane transporter proteins. Here we present the three-dimensional structure at 6.5 Å resolution of a bacterial member of this superfamily, OxlT. The structure, derived from an electron crystallographic analysis of two-dimensional crystals, reveals that the 12 helices in the OxlT molecule are arranged around a central cavity, which is widest at the center of the membrane. The helices divide naturally into three groups: a peripheral set comprising helices 3, 6, 9 and 12; a second set comprising helices 2, 5, 8 and 11 that faces the central substrate transport pathway across most of the length of the membrane; and a third set comprising helices 1, 4, 7 and 10 that participate in the pathway either on the cytoplasmic side (4 and 10) or on the periplasmic side (1 and 7). Overall, the architecture of the protein is remarkably symmetric, providing a compelling molecular explanation for the ability of such transporters to carry out bi-directional substrate transport.

OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation

The dynamin-related GTPase OPA1 is mutated in autosomal dominant optic atrophy (DOA) (Kjer type), an inherited neuropathy of the retinal ganglion cells. OPA1 is essential for the fusion of the inner mitochondrial membranes, but its mechanism of action remains poorly understood. Here we show that OPA1 has a low basal rate of GTP hydrolysis that is dramatically enhanced by association with liposomes containing negative phospholipids such as cardiolipin. Lipid association triggers assembly of OPA1 into higher order oligomers. In addition, we find that OPA1 can promote the protrusion of lipid tubules from the surface of cardiolipin-containing liposomes. In such lipid protrusions, OPA1 assemblies are observed on the outside of the lipid tubule surface, a protein-membrane topology similar to that of classical dynamins. The membrane tubulation activity of OPA1 is suppressed by GTPγS. OPA1 disease alleles associated with DOA display selective defects in several activities, including cardiolipin association, GTP hydrolysis and membrane tubulation. These findings indicate that interaction of OPA1 with membranes can stimulate higher order assembly, enhance GTP hydrolysis and lead to membrane deformation into tubules.

Structural Model for 12-Helix Transporters Belonging to the Major Facilitator Superfamily

The major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans. We have recently reported the three-dimensional structure, at 6.5 A resolution, of the oxalate transporter, OxlT, a representative member of this superfamily. In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane. Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins. Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily.

3D Imaging of mammalian cells with ion-abrasion scanning electron microscopy

Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and melanocytes at resolutions as high as approximately 6 and approximately 20 nm in the directions parallel and perpendicular, respectively, to the direction of ion-beam milling. The 3D images demonstrate the striking spatial relationships between specific organelles such as mitochondria and membranes of the endoplasmic reticulum, and the distribution of unique cellular components such as melanosomes. We also show that 10nm-sized gold particles and quantum dot particles with 7 nm-sized cores can be detected in single cross-sectional images. IA-SEM is thus a useful tool for imaging large mammalian cells in their entirety at resolutions in the nanometer range.

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at −150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.

Dynamins at a glance.

The superfamily of dynamins includes classical dynamins and dynamin-related proteins. Classical dynamins are proteins that share sequence similarity with the first described dynamin, which is a large GTPase with five characteristic domains. Dynamin-related proteins differ from classical dynamins in

Correlative 3D imaging of Whole Mammalian Cells with Light and Electron Microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10-20nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

Projection structure and molecular architecture of OxlT, a bacterial membrane transporter.

The major facilitator superfamily (MFS) represents the largest collection of evolutionarily related members within the class of membrane ‘carrier’ proteins. OxlT, a representative example of the MFS, is an oxalate‐transporting membrane protein in Oxalobacter formigenes. From an electron crystallographic analysis of two‐dimensional crystals of OxlT, we have determined the projection structure of this membrane transporter. The projection map at 6 Å resolution indicates the presence of 12 transmembrane helices in each monomer of OxlT, with one set of six helices related to the other set by an approximate internal two‐fold axis. The projection map reveals the existence of a central cavity, which we propose to be part of the pathway of oxalate transport. By combining information from the projection map with related biochemical data, we present probable models for the architectural arrangement of transmembrane helices in this protein superfamily.

APOL1 Kidney Disease Risk Variants: An Evolving Landscape

Apolipoprotein L1 (APOL1) genetic variants account for much of the excess risk of chronic and end-stage kidney disease, which results in a significant global health disparity for persons of African ancestry. We estimate the lifetime risk of kidney disease in APOL1 dual-risk allele individuals to be at least 15%. Experimental evidence suggests a direct role of APOL1 in pore formation, cellular injury, and programmed cell death in renal injury. The APOL1 BH3 motif, often associated with cell death, is unlikely to play a role in APOL1-induced cytotoxicity because it is not conserved within the APOL family and is dispensable for cell death in vitro. We discuss two models for APOL1 trypanolytic activity: one involving lysosome permeabilization and another involving colloid-osmotic swelling of the cell body, as well as their relevance to human pathophysiology. Experimental evidence from human cell culture models suggests that both mechanisms may be operative. A systems biology approach whereby APOL1-associated perturbations in gene and protein expression in affected individuals are correlated with molecular pathways may be productive to elucidate APOL1 function in vivo.

Therapeutics for APOL1 nephropathies: putting out the fire in the podocyte

APOL1 nephropathies comprise a range of clinical and pathologic syndromes, which can be summarized as focal segmental glomerulosclerosis, in various guises, and arterionephrosclerosis, otherwise known as hypertensive kidney diseases. Current therapies for these conditions may achieve therapeutic targets, reduction in proteinuria and control of blood pressure, respectively, but often fail to halt the progressive decline in kidney function. It appears that current therapies fail to address certain underlying critical pathologic processes that are driven, particularly in podocytes and microvascular cells, by the APOL1 renal risk genetic variants. Mechanisms hypothesized to be responsible for APOL1 variant-associated cell injury can be summarized in five domains: increased APOL1 gene expression, activation of inflammasomes, activation of protein kinase R, electrolyte flux across plasma or intracellular membranes, and altered endolysosomal trafficking associated with endoplasmic reticulum stress. We briefly review the available evidence for these five mechanisms and suggest possible novel therapeutic approaches.