Valverde et al. show that the autophagy protein ATG2 functions in autophagosome biogenesis by transferring lipids at ER–autophagosome contact sites.

BackgroundPeptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood.ResultsThe X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1’s substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter.ConclusionThis study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0167-8) contains supplementary material, which is available to authorized users.

Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Inhibitors for membrane transporters have been shown to be indispensable as drugs and tool compounds. The proton-dependent oligopeptide transporters PEPT1 and PEPT2 from the SLC15 family play important roles in human and mammalian physiology. With Lys[Z(NO2)]-Val (LZNV), a modified Lys-Val dipeptide, a potent transport inhibitor for PEPT1 and PEPT2 is available. Here we present the crystal structure of the peptide transporter YePEPT in complex with LZNV. The structure revealed the molecular interactions for inhibitor binding and a previously undescribed mostly hydrophobic pocket, the PZ pocket, involved in interaction with LZNV. Comparison with a here determined ligand-free structure of the transporter unveiled that the initially absent PZ pocket emerges through conformational changes upon inhibitor binding. The provided biochemical and structural information constitutes an important framework for the mechanistic understanding of inhibitor binding and action in proton-dependent oligopeptide transporters.

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.

A typical feature of type II restriction endonucleases (REases) is their obligate sequence specificity and requirement for Mg(2+) during catalysis. R.KpnI is an exception. Unlike most other type II REases, the active site of this enzyme can accommodate Mg(2+), Mn(2+), Ca(2+), or Zn(2+) and cleave DNA. The enzyme belongs to the HNH superfamily of nucleases and is characterized by the presence of a ββα-Me finger motif. Residues D148, H149, and Q175 together form the HNH active site and are essential for Mg(2+) binding and catalysis. The unique ability of the enzyme to cleave DNA in the presence of different metal ions is exploited to generate mutants that are specific to one particular metal ion. We describe the generation of a Mn(2+)-dependent sequence specific endonuclease, defective in DNA cleavage with Mg(2+) and other divalent metal ions. In the engineered mutant, only Mn(2+) is selectively bound at the active site, imparting Mn(2+)-mediated cleavage. The mutant is impaired in concerted double-stranded DNA cleavage, leading to accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altered reaction conditions. The active site fluidity of REases allowing flexible accommodation of catalytic cofactors thus forms a basis for engineering selective metal ion-dependent REase additionally possessing nicking activity.

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.