Wnt signaling pathways regulate many developmental responses; however, little is known about how Wnt ligands function on a biochemical level. Recent studies have shown that Wnt-3a is palmitoylated before secretion. Here we report that Drosophila Wnt-1 (Wingless) also undergoes a lipid modification. Lipidation occurs in the endoplasmic reticulum and is dependent on Porcupine, a putative O-acyltransferase. After modification, DWnt-1 partitions as a membrane-anchored protein and is sorted into lipid raft detergent-insoluble microdomains. Lipidation, raft targeting, and secretion can be blocked by the addition of 2-bromopalmitate, a competitive inhibitor of O-acyltransferase activity. Based on these results we propose a model whereby lipidation targets Wnt-1 to secretory vesicles that deliver the ligand to specialized microdomains at the cell surface where it can be packaged for secretion.

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This

Lipid-protein interactions, critical for the folding, stability and function of membrane proteins, can be both of mechanical and chemical nature. Mechanical properties of lipid systems can be suitably influenced by physical factors so as to facilitate membrane protein folding. We demonstrate here that by modulating lipid dynamics transiently using heat, rapid folding of two 8-stranded transmembrane β-barrel proteins OmpX and OmpA1–171, in micelles and vesicles, can be achieved within seconds. Folding kinetics using this ‘heat shock’ method shows a dramatic ten to several hundred folds increase in refolding rate along with ~100% folding efficiency. We establish that OmpX thus folded is highly thermostable even in detergent micelles, and retains structural characteristics comparable to the protein in bilayers.

Cyclic AMP (cAMP)-dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1) share several cellular proteins as substrates. However, to date no other similarities between the two kinases or interactions between them have been reported. Here, we describe novel interactions between subunits of PKA and RSK1 that are dependent upon the activation state of RSK1 and determine its subcellular distribution and biological actions. Inactive RSK1 interacts with the type I regulatory subunit (RI) of PKA. Conversely, active RSK1 interacts with the catalytic subunit of PKA (PKAc). Binding of RSK1 to RI decreases the interactions between RI and PKAc, while the binding of active RSK1 to PKAc increases interactions between PKAc and RI and decreases the ability of cAMP to stimulate PKA. The RSK1/PKA subunit interactions ensure the colocalization of RSK1 with A-kinase PKA anchoring proteins (AKAPs). Disruption of the interactions between PKA and AKAPs decreases the nuclear accumulation of active RSK1 and, thus, increases its cytosolic content. This subcellular redistribution of active RSK1 is manifested by increased phosphorylation of its cytosolic substrates tuberous sclerosis complex 2 and BAD by epidermal growth factor along with decreased cellular apoptosis.Cyclic AMP (cAMP)-dependent protein kinase (PKA) regulates a wide variety of metabolic and functional processes, including cell proliferation, actin cytoskeleton rearrangements, and gene transcription (41). PKA is a tetramer that consists of two regulatory (R) and two catalytic (PKAc) subunits in its inactive form. Binding of cAMP to the R subunits of PKA results in the dissociation of the PKAc subunits, which can then phosphorylate their substrate proteins. The PKA holoenzyme is localized in the proximity of its target proteins by A-kinase PKA anchoring proteins (AKAPs) that act as scaffolds (2, 52). Moreover, by binding to additional signaling molecules, the AKAPs facilitate the coordination and integration of several signals to regulate biological events (2, 52). The two main forms of the PKA regulatory subunits, RI and RII, have different affinities for the various AKAPs. Although most of the AKAPs appear to have a higher affinity for RII subunits (2), certain AKAPs such as D-AKAP1 and D-AKAP2 preferentially bind the RI subunit (22-24).In addition to AKAPs, other well-characterized protein interactions are involved in the intracellular targeting and regulation of PKA and its subunits. For instance, RI␣ associates with cytochrome c oxidase subunit Vb and increases its activity whereas exposure to cAMP inhibits cytochrome c oxidase activity and releases cytochrome c from mitochondria and activates apoptosis (55). PAP7, a protein involved in hormonal regulation of steroid formation, also binds RI␣ at the outer mitochondrial membrane and helps in cholesterol uptake and transport to the inner mitochondrial membrane (28). Likewise, Rab32, a small GTP binding protein, can act as an AKAP for the RII subunits and target PKA to mitochondria (3). In addition, the amount of RI...

The biogenesis of transmembrane β-barrels (outer membrane proteins, or OMPs) is an elaborate multistep orchestration of the nascent polypeptide with translocases, barrel assembly machinery, and helper chaperone proteins. Several theories exist that describe the mechanism of chaperone-assisted OMP assembly in vivo and unassisted (spontaneous) folding in vitro. Structurally, OMPs of bacterial origin possess even-numbered strands, while mitochondrial β-barrels are even- and odd-stranded. Several underlying similarities between prokaryotic and eukaryotic β-barrels and their folding machinery are known; yet, the link in their evolutionary origin is unclear. While OMPs exhibit diversity in sequence and function, they share similar biophysical attributes and structure. Similarly, it is important to understand the intricate OMP assembly mechanism, particularly in eukaryotic β-barrels that have evolved to perform more complex functions. Here, we deliberate known facets of β-barrel evolution, folding, and stability, and attempt to highlight outstanding questions in β-barrel biogenesis and proteostasis.