Retromer forms low order oligomers on supported lipid bilayers.

Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)-retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (∼18%), dimer (∼35%), trimer (∼24%), and tetramer (∼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.

Dodecyl-ß-melibioside Detergent Micelles as a Medium for Membrane Proteins.

There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-ß-melibioside (ß-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the ß-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-ß-maltoside (ß-DDM). ß-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into ß-DDMB yielded activities that were 40% higher than those of DAGK purified into ß-DDM. ß-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. ß-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.

Structural and biochemical differences between the Notch and the amyloid precursor protein transmembrane domains.

γ-Secretase cleavage of the Notch receptor transmembrane domain is a critical signaling event for various cellular processes. Efforts to develop inhibitors of γ-secretase cleavage of the amyloid-ß precursor C99 protein as potential Alzheimer's disease therapeutics have been confounded by toxicity resulting from the inhibition of normal cleavage of Notch. We present biochemical and structural data for the combined transmembrane and juxtamembrane Notch domains (Notch-TMD) that illuminate Notch signaling and that can be compared and contrasted with the corresponding traits of C99. The Notch-TMD and C99 have very different conformations, adapt differently to changes in model membrane hydrophobic span, and exhibit different cholesterol-binding properties. These differences may be exploited in the design of agents that inhibit cleavage of C99 while allowing Notch cleavage.

Notch Transmembrane Domain: Secondary Structure and Topology.

The Notch signaling pathway is critical in development, neuronal maintenance, and hematopoiesis. An obligate step in the activation of this pathway is cleavage of its transmembrane (TM) domain by γ-secretase. While the soluble domains have been extensively studied, little has been done to characterize its TM and flanking juxtamembrane (JM) segments. Here, we present the results of nuclear magnetic resonance (NMR) studies of the human Notch1 TM/JM domain. The TM domain is largely α-helical. While the flanking JM segments do not adopt regular secondary structure, they interact with the membrane surface, suggesting membrane interactions may play a role in modulating its cleavage by γ-secretase and subsequent NOTCH signaling function.

Tailoring of membrane proteins by alternative splicing of pre-mRNA.

Alternative splicing (AS) of RNA is a key mechanism for diversification of the eukaryotic proteome. In this process, different mRNA transcripts can be produced through altered excision and/or inclusion of exons during processing of the pre-mRNA molecule. Since its discovery, AS has been shown to play roles in protein structure, function, and localization. Dysregulation of this process can result in disease phenotypes. Moreover, AS pathways are promising therapeutic targets for a number of diseases. Integral membrane proteins (MPs) represent a class of proteins that may be particularly amenable to regulation by alternative splicing because of the distinctive topological restraints associated with their folding, structure, trafficking, and function. Here, we review the impact of AS on MP form and function and the roles of AS in MP-related disorders such as Alzheimer's disease.

Purification and characterization of the human γ-secretase activating protein.

Alzheimer's disease is a fatal neurological disorder that is a leading cause of death, with its prevalence increasing as the average life expectancy increases worldwide. There is an urgent need to develop new therapeutics for this disease. A newly described protein, the γ-secretase activating protein (GSAP), has been proposed to promote elevated levels of amyloid-ß production, an activity that seems to be inhibited using the well-establish cancer drug, imatinib (Gleevec). Despite much interest in this protein, there has been little biochemical characterization of GSAP. Here we report protocols for the recombinant bacterial expression and purification of this potentially important protein. GSAP is expressed in inclusion bodies, which can be solubilized using harsh detergents or urea; however, traditional methods of refolding were not successful in generating soluble forms of the protein that contained well-ordered and homogeneous tertiary structure. However, GSAP could be solubilized in detergent micelle solutions, where it was seen to be largely α-helical but to adopt only heterogeneous tertiary structure. Under these same conditions, GSAP did not associate with either imatinib or the 99-residue transmembrane C-terminal domain of the amyloid precursor protein. These results highlight the challenges that will be faced in attempts to manipulate and characterize this protein.