Abstract
The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMPs) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, is the entry point for clients to the guided entry of tail-anchored protein (GET) pathway, which targets endoplasmic reticulum-bound TA IMPs. Consisting of three structurally independent domains, the C terminus of Sgt2 binds to the hydrophobic transmembrane domain (TMD) of clients. However, the exact binding interface within Sgt2 and molecular details that underlie its binding mechanism and client preference are not known. Here, we reveal the mechanism of Sgt2 binding to hydrophobic clients, including TA IMPs. Through sequence analysis, biophysical characterization, and a series of capture assays, we establish that the Sgt2 C-terminal domain is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove approximately 15 Å long that is consistent with our observed higher affinity for client TMDs with a hydrophobic face and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing cochaperone proteins, demonstrating structural and sequence-based similarities to the DP domains in the yeast Hsp90 and Hsp70 coordinating protein, Sti1.
Highlights
An inherently complicated problem of cellular homeostasis is the biogenesis of hydrophobic integral membrane proteins (IMPs), which are synthesized in the cytoplasm and must be targeted and inserted into a lipid bilayer
Homologs of Saccharomyces cerevisiae Sgt2 and Homo sapiens small glutamine tetratricopeptide repeat protein alpha (SGTA) are involved in a variety of cellular processes regarding the homeostasis of membrane proteins including the targeting of TA IMPs [9, 12,13,14], retrograde transport of membrane proteins for ubiquitination and subsequent proteasomal degradation [15], and regulation of mislocalized membrane proteins (MLPs) [16, 17]
The N-terminal domain forms a homodimer composed of a four-helix bundle with twofold symmetry that primarily binds to the ubiquitin-like domain (UBL) of Get5/ Ubl4A for TA IMP targeting [36, 40] or interacts with the UBL on the N-terminal region of Bag6 [41] where it is thought to initiate downstream degradation processes [15, 28, 29]
Summary
Based on sequence alignment (Fig. 1A), the Sgt2-C contains a conserved core of six predicted helices flanked by unstructured loops that vary in length and sequence. For all of the helices, alanine replacement of the hydrophobic residues significantly reduces binding of Sbh to Sgt2-C (Fig. 2, E and F) While these mutants expressed at similar levels to the wild-type (WT) sequence, one cannot rule out that some of these changes may affect the tertiary structure of this domain. Based on the six conserved α-helical amphipathic segments (Fig. 1A) that contain hydrophobic residues critical for client binding (Fig. 2, D and E), we expect some folded structure to exist. To correct for this, we replaced the sequence of the N-terminal loop of hSgt2-C with the ySgt2-C loop and ran structure prediction with the pairwise distance restraints This resulted in a model where the loop occupies the groove and, when pruned away, suggests the hydrophobic hand seen in yeast (Fig. S5C, middle boxed).
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