Abstract
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.
Highlights
The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras
Several SOS constructs were fused with an enhanced green fluorescence protein (EGFP) tag at the C terminus to enable single-molecule visualization
These results demonstrate that fulllength SOS constructs are successfully expressed in HEK293T cells and are monomeric in lysates
Summary
Characterization of SOS expressed in HEK293T cells. A series of SOS constructs, including the native, full-length protein were transiently expressed in HEK293T cells and collected in crude, whole-cell lysates (Fig. 1a). Traces of fluorescence from single EGFP-tagged SOSFL molecules immobilized on a glass substrate exhibited predominantly single step photobleaching, consistent with the monomeric state (Fig. 1d,e) These results demonstrate that fulllength SOS constructs are successfully expressed in HEK293T cells and are monomeric in lysates. The membrane-tethered Ras and SOS were laterally confined in an array of micrometre-scale supported lipid bilayers (SLB) that are partitioned by nanofabricated chromium metal lines (Fig. 2a)[47,48,49]. In this assay, SOS constructs (without the EGFP tag) in cell lysate were incubated with the Ras-coupled bilayers. A single SOS catalyses the exchange of fluorescent GTP-bound a
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