Structural investigations of the Get4/Get5/Sgt2 complex

Abstract

Tail-anchored (TA) proteins are classified by a single transmembrane helix at the extreme carboxyl terminus. In yeast, the Guided Entry of Tail-anchored proteins (GET) pathway facilitates the post-translational targeting of TA proteins to the endoplasmic reticulum (ER). A complex consisting of Get4, Get5, Sgt2 and chaperones delivers ER destined substrates to Get3, which then targets the substrate to the ER membrane. We have structurally characterized the Get4/Get5/Sgt2 complex. Get4 is an alpha-helical repeat protein that directly binds Get3. Get4 and Get5 form an obligate dimer mediated by the amino terminal domain of Get5, and this heterodimer forms a higher order dimer mediated by the carboxyl terminal domain of Get5. We have determined structures of the Get5 homodimerization domain by crystallography and solution NMR and found it is a novel, high-affinity oligomerization motif. Sgt2 contains a tetratricopeptide repeat (TPR) domain that binds to a wide variety of heat shock cognate (HSC) protein families. A crystal structure of the TPR domain with an HSC-analog provides an explanation for this low specificity. Sgt2 also contains an amino terminal homodimerization domain that additionally binds a ubiquitin-like domain within Get5. We utilize bioSAXS to combine high-resolution structural data, generating a model of the Get4/Get5/Sgt2 complex in solution.

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