The folding of K K-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable K K-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure. ß

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue, and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs, which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD–LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.DOI: http://dx.doi.org/10.7554/eLife.07485.001

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Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.plant | endoplasmic reticulum | reticulon | amphipathic helix | membrane curvature A s the gateway to the secretory pathway, the endoplasmic reticulum (ER) is responsible for secretory protein translocation, folding, and quality control and is thus central to the maintenance of cellular homeostasis (1). In plant cells, the ER consists of the nuclear envelope and a dynamic peripheral network of cisternae and, more predominantly, tubules extending throughout the cytoplasm and across cell boundaries through plasmodesmata. Several proteins have been implicated in the shaping of the ER membrane. In plants, these include ROOT HAIR DEFECTIVE3 (RHD3), which is orthologous to mammalian atlastins and yeast Sey1p and is likely important for the formation of three-way junctions (2, 3), and the proteins of the reticulon (RTN) family. The RTNs are preferentially associated with ER tubules and the curved edges of cisternae. When overexpressed in planta, RTNs induce severe constrictions of ER tubules and are able to convert ER membrane sheets into tubules (4-6).The mechanism by which RTNs generate and/or stabilize curvature of a membrane has been attributed to the reticulon homology domain (RHD): a conserved domain of ∼200 amino acids containing two long hydrophobic regions flanking a hydrophilic loop. These two hydrophobic regions can each be further subdivided into two transmembrane domains (TMDs), resulting in a W-like topology. The RHD is also found in the DP1 (deleted in polyposis) family of proteins that includes Yop1p in yeast and human REEPs (receptor expression-enhancing proteins). The four hydrophobic TMDs of the plant RHD are proposed to form wedge-like hairpins in the lipid bilayer, which, in combination with the RHD-mediated oligomerization of RTNs into low-mobility oligomers, appear to be sufficient to induc...

The use of the computer program CONTIN to analyze pulsed-field gradient NMR (PFG-NMR) data for several standard humic and fulvic acids is described. An advantage of PFG-NMR analysis is that integration of different spectral regions provides a picture of how the diffusion coefficients vary with functional group composition for a given sample. Using prior knowledge of the sample and the principle of parsimony, CONTIN approximates a solution to the inverse Laplace transform applied to the decay of peak intensity with gradient area in the PFG-NMR experiment. Thus, a continuous distribution of diffusion coefficients is resolved for the polydisperse humic and fulvic acids. The results of the CONTIN analyses are in the form of a distribution function and a two-dimensional DOSY plot. The 2D DOSY spectrum displays chemical shifts along one axis and diffusion coefficients along the other, while a number-average diffusion coefficient, D(N), a weight-average diffusion coefficient, D(W), and a most probable diffusion coefficient, D(P), are realized from the diffusion coefficient distribution. For all spectral regions of each humic sample, D(W) was greater than D(N), which in turn was greater than or equal to the D(P), suggesting that the diffusion coefficient distribution is weighted toward smaller, more rapidly diffusing molecules. Polydispersities, estimated from the ratio D(W)/D(N), were less than the reported M(W)/M(N) values for similar humic substances. Thus, the D(W)/D(N) ratio obtained by CONTIN analysis of PFG-NMR data can be at least a qualitative, and at best a semiquantitative, indication of the polydispersity of the humic sample, but should not be used as a quantitative measure of polydispersity.

Background: Major histocompatibility complex class II molecules are structurally and functionally heterogeneous. Results: Combined mutagenesis and structural studies establish a role for pairing between conserved transmembrane (TM) GXXXG dimerization motifs in determining class II conformation. Conclusion: Differential pairing of highly conserved TM domain dimerization motifs contributes to class II structure and function. Significance: Global conformation contributes to the function of peptide-class II complexes.

The Major Histocompatibility Complex Class II (Class II MHC) and invariant chain (Ii) proteins are key initiators of an immune response to invading pathogens. Following biosynthesis, three MHCalpha/beta hetero-dimers associate with an Ii homotrimer to form a nine-chain protein complex. Only as part of this complex are the MHC molecules exported to the cell surface to trigger an immune response. Previous reports implicate the transmembrane (TM) domains of all three proteins in correct assembly, ligand binding and function of Class II MHC. Building on our previous work that revealed the Ii TM domain may contribute significantly to correct assembly of the full-length protein, we have used a variety of genetic, biophysical and computational methods to investigate the role of the TM domains in stabilizing MHCalpha/beta heterodimers. Using the in vivo GALLEX assay, we find that the TM domains of both proteins form strong homo- and hetero-oligomers in natural membranes that are stabilized by GXXXG motifs within the sequence. Förster resonance energy transfer (FRET) measurements, using fluorescently-tagged peptides derived from the TM domains of each protein, were then employed to confirm the presence of TM helix-helix hetero-interactions in detergent micelles, as well as the stoichiometry of these interactions. Our results are summarized in a revised model of Class II MHC-Ii complex formation that illustrates key protein-protein contacts. This work provides the first evidence that the TM domains of the Class II MHC molecules are capable of significant protein-protein interactions that may help to stabilize or even initiate formation of the MHC-Ii complex.

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.