Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast

Abstract

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

Capture and delivery of tail-anchored proteins to the endoplasmic reticulum

“…In vitro reconstitution confirmed a high-affinity interaction between Get4-Get5 and ribosomes, and protein–protein and protein–RNA cross-linking analyses pinpointed the polypeptide exit tunnel of the ribosome as the Get4-Get5 binding site ( Fig. 1 ; Zhang et al, 2021 ). Get4-Get5 bridge interactions between Sgt2 and Get3 to facilitate handover of the TA protein to the downstream chaperone, and therefore functional significance of Get4-Get5 ribosome binding must be coupled to TA protein capture by Sgt2.…”

“…Get4-Get5 bridge interactions between Sgt2 and Get3 to facilitate handover of the TA protein to the downstream chaperone, and therefore functional significance of Get4-Get5 ribosome binding must be coupled to TA protein capture by Sgt2. Indeed, it was recently shown that Get4-Get5 act as a binding platform for recruitment of Sgt2 to ribosomes and that the presence of Get4-Get5 on ribosomes enhances TA protein capture by Sgt2 ( Zhang et al, 2021 ). Crystal structures of GET pathway subcomplexes demonstrate that the N-terminal domains of the Sgt2 homodimer interact with the central UBL domain of Get5, while the N-terminal region of Get5 mediates interaction with C-terminal region of Get4 ( Chang et al, 2010 ; Chartron et al, 2012a ; Simon et al, 2013 ).…”

“…The discovery of ribosome-associated populations of the GET pretargeting complex and the BAG6 complex and SGTA in yeast and mammals, respectively, raises the question of how these complexes, which are significantly less abundant than cytosolic ribosomes, are able to identify ribosomes synthesizing TA proteins. Analogous to SRP, which probes the translating ribosome population, preferentially binding ribosomes translating proteins with a signal sequence ( Berndt et al, 2009 ; Holtkamp et al, 2012 ; Ogg and Walter, 1995 ; Voorhees and Hegde, 2015 ), yeast Get4-Get5 show increased association with RNCs containing a TA/TMD in the exit tunnel ( Zhang et al, 2021 ). Mammalian SGTA is likewise selectively recruited to ribosomes synthesizing membrane proteins ( Leznicki and High, 2020 ).…”

Protein Targeting Into the Thylakoid Membrane Through Different Pathways

“…The posttranslational insertion of TA proteins from the cytosol to cellular membranes depends on the GET pathway in yeast and the TRC (Transmembrane Recognition Complex) pathway in mammals ( Farkas et al, 2019 ; Farkas and Bohnsack, 2021 ). The yeast cytosolic GET machinery includes a pretargeting complex consisting of Sgt2, a small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (SGTA in mammals), Get4 (TRC35 in mammals), and Get5 (UBL4 in mammals), of which Sgt2 captures newly synthesized TA proteins from the ribosomal exit tunnel and is considered the most upstream factor in the GET pathway ( Wang et al, 2010 ; Shao et al, 2017 ; Zhang et al, 2021 ). Subsequently Get4 and Get5 build up a “scaffolding complex” by recruiting Get3 and the Sgt2-TA complex, respectively.…”

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

“…Interestingly, yeast lacks a homolog of BAG6 with consequences for the hierarchical organization of the GET pathway compared to mammalian cells. Specifically, Get4/5 (GET4/5 homolog) binds the yeast ribosome overlapping with SRP via the Get5 subunit [ 250 ]. Sgt2 (SGTA homolog) is recruited to the tunnel exit only in a subsequent step.…”