Membrane anchoring facilitates colocalization of enzymes in plant cytochrome P450 redox systems.

Plant metabolism depends on cascade reactions mediated by dynamic enzyme assemblies known as metabolons. In this context, the cytochrome P450 (P450) superfamily catalyze key reactions underpinning the unique diversity of bioactive compounds. In contrast to their soluble bacterial counterparts, eukaryotic P450s are anchored to the endoplasmic reticulum membrane and serve as metabolon nucleation sites. Hence, membrane anchoring appears to play a pivotal role in the evolution of complex biosynthetic pathways. Here, a model membrane assay enabled characterization of membrane anchor dynamics by single molecule microscopy. As a model system, we reconstituted the membrane anchor of cytochrome P450 oxidoreductase (POR), the ubiquitous electron donor to all microsomal P450s. The transmembrane segment in the membrane anchor of POR is relatively conserved, corroborating its functional importance. We observe dynamic colocalization of the POR anchors in our assay suggesting that membrane anchoring might promote intermolecular interactions and in this way impact assembly of metabolic multienzyme complexes.

A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS.

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) is a key Ras activator that is autoinhibited in the cytosol and activates upon membrane recruitment. Autoinhibition release involves structural rearrangements of the protein at the membrane and thus introduces a delay between initial recruitment and activation. In this study, we designed a single-molecule assay to resolve the time between initial receptor-mediated membrane recruitment and the initiation of GEF activity of individual SOS molecules on microarrays of Ras-functionalized supported membranes. The rise-and-fall shape of the measured SOS activation time distribution and the long mean time scale to activation (~50 seconds) establish a basis for kinetic proofreading in the receptor-mediated activation of Ras. We further demonstrate that this kinetic proofreading is modulated by the LAT (linker for activation of T cells)-Grb2-SOS phosphotyrosine-driven phase transition at the membrane.

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

Covalent Ras Dimerization on Membrane Surfaces through Photosensitized Oxidation.

Ras, a small GTPase found primarily on the inner leaflet of the plasma membrane, is an important signaling node and an attractive target for anticancer therapies. Lateral organization of Ras on cellular membranes has long been a subject of intense research; in particular, whether it forms dimers on membranes as part of its regulatory function has been a point of great interest. Here we report Ras dimer formation on membranes by Type II photosensitization reactions, in which molecular oxygen mediates the radicalization of proteins under typical fluorescence experimental conditions. The presence of Ras dimers on membranes was detected by diffusion-based fluorescence techniques including fluorescence correlation spectroscopy and single particle tracking, and molecular weights of the stable covalently coupled species were confirmed by gel electrophoresis. Fluorescence spectroscopy implicates interprotein dityrosine as one of the dimerization motifs. The specific surface tyrosine distribution on Ras renders the protein especially sensitive to this reaction, and point mutations affecting surface tyrosines are observed to alter dimerization potential. The photosensitization reactions are reflective of physiological oxidative stress induced by reactive oxygen species, suggesting such processes may occur naturally and influence signaling pathways in cells.