Geranylgeranyl switching regulates GDI-Rab GTPase recycling

Rab GTPases, key regulators of membrane targeting and fusion, require the covalent attachment of geranylgeranyl lipids to their C terminus for function. To elucidate the role of lipid in Rab recycling, we have determined the crystal structure of Rab guanine nucleotide dissociation inhibitor (alphaGDI) in complex with a geranylgeranyl (GG) ligand (H(2)N-Cys-(S-GG)-OMe). The lipid is bound beneath the Rab binding platform in a shallow hydrophobic groove. Mutation of the binding pocket in the brain-specific alphaGDI leads to mental retardation. Strikingly, lipid binding acts through a conserved allosteric switching mechanism to promote release of the GDI-Rab[GDP] complex from the membrane.     

Amyloid as a natural product

Amyloid fibrils, such as those found in Alzheimer's and the gelsolin amyloid diseases, result from the misassembly of peptides produced by either normal or aberrant intracellular proteolytic processing. A paper in this issue by Marks and colleagues (Berson et al., 2003) demonstrates that intra-melanosome fibrils are formed through normal biological proteolytic processing of an integral membrane protein. The resulting peptide fragment assembles into fibrils promoting the formation of melanin pigment granules. These results, along with the observation that amyloid fibril formation by bacteria is highly orchestrated, suggest that fibril formation is an evolutionary conserved biological pathway used to generate natural product nanostructures.     

Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease

Choroideremia is an X-chromosome-linked disease that leads to the degeneration of the choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye. The gene product defective in choroideremia, CHM, is identical to Rab escort protein 1 (REP1). CHM/REP1 is an essential component of the catalytic geranylgeranyltransferase II complex (GGTrII) that delivers newly synthesized small GTPases belonging to the RAB gene family to the catalytic complex for post-translational modification. CHM/REP family members are evolutionarily related to members of the guanine nucleotide dissociation inhibitor (GDI) family, proteins involved in the recycling of Rab proteins required for vesicular membrane trafficking through the exocytic and endocytic pathways, forming the GDI/CHM superfamily. Biochemical and structural analyses have now revealed a striking parallel in the organization and function of these two families allowing us to generate a general model for GDI/CHM superfamily function in health and disease.     

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.     

Ligand connected metal clusters. The molecular structures and solid state packing of {Ru3(CO)11}2(bis(diphenylphosphino)ethane) and {Ru3(CO)11}2(1,4-bis(diphenylphosphino)benzene)

The preparation and structural characterization of {Ru₃(CO)₁₁}₂(1,4-bis(diphenylphosphino)benzene), a modified synthesis of 1,4-bis(diphenylphosphino)benzene, and the structural characterization of {Ru₃(CO)₁₁}₂(bis(diphenylphosphino)ethane) are reported. In both compounds two metal cluster units are connected through ditertiary-phosphine ligands. Both molecules consist of centrosymmetric units in which the diphosphine ligands are largely covered by the triangular ruthenium clusters. No direct interaction between the two cluster units occurs within individual molecules. Molecular packing in the solid state is dominated by interactions between sets of carbon monoxide ligands in motifs that were previously identified in the solid state structure of the parent cluster, Ru₃(CO)₁₂.     

STUDIES OF NITRATE TRANSPORT BY MARINE PHYTOPLANKTON USING 36Cl-ClO3− AS A TRANSPORT ANALOGUE. I. PHYSIOLOGICAL FINDINGS1

Transport of a nitrate analogue, ³⁶Cl-ClO₃⁻, was examined in two diatoms, Skeletonema costatum (Greve.) Cleve and Nitzschia closterium (Ehrenb) W. Sm. A dinoflagellate, Gonyaulax polyedra did not transport ClO₃⁻. Transport of ³⁶Cl-ClO₃⁻ by diatoms appeared to be active and showed saturation kinetics. The data were fitted by Michaelis-Menten equation at all but the lowest chlorate concentrations (where plots of S vs. v showed a slight concave bend). Affinity of cells for nitrate was considerably higher than for chlorate. The K_i for nitrate inhibition of chlorate transport was calculated assuming competitive inhibition. Light had little or no effect on chlorate transport. Pulse-chase experiments demonstrated that (1) ClO₃⁻ (hence NO₃⁻) was stored in two intracellular compartments of equal size, (2) internal ClO₃⁻ was exchangeable with external ClO₃⁻ (rates of efflux and influx were measured), and (3) efflux of intracellular ClO₃⁻ showed transient states following a chase of ClO₃⁻ or NO₃⁻ which stabilized after 10–20 min. Transport of chlorate was a function of growth phase.     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.     

An adaptable standard for protein export from the endoplasmic reticulum

To provide an integrated view of endoplasmic reticulum (ER) function in protein export, we have described the interdependence of protein folding energetics and the adaptable biology of cellular protein folding and transport through the exocytic pathway. A simplified treatment of the protein homeostasis network and a formalism for how this network of competing pathways interprets protein folding kinetics and thermodynamics provides a framework for understanding cellular protein trafficking. We illustrate how folding and misfolding energetics, in concert with the adjustable biological capacities of the folding, degradation, and export pathways, collectively dictate an adaptable standard for protein export from the ER. A model of folding for export (FoldEx) establishes that no single feature dictates folding and transport efficiency. Instead, a network view provides insight into the basis for cellular diversity, disease origins, and protein homeostasis, and predicts strategies for restoring protein homeostasis in protein-misfolding diseases.     

Functional amyloid--from bacteria to humans

Amyloid--a fibrillar, cross beta-sheet quaternary structure--was first discovered in the context of human disease and tissue damage, and was thought to always be detrimental to the host. Recent studies have identified amyloid fibers in bacteria, fungi, insects, invertebrates and humans that are functional. For example, human Pmel17 has important roles in the biosynthesis of the pigment melanin, and the factor XII protein of the hemostatic system is activated by amyloid. Functional amyloidogenesis in these systems requires tight regulation to avoid toxicity. A greater understanding of the diverse physiological applications of this fold has the potential to provide a fresh perspective for the treatment of amyloid diseases.