Integrating the results presented here, we propose that the Get3 ATPase has a second cellular function in addition to its well-characterized role as a factor that targets newly synthesized tail-anchored proteins to insertion sites in the ER membrane (). This second function as a holdase exploits the chaperone capacity of Get3 to bind hydrophobic regions of proteins and becomes relevant under conditions that interfere with the tail-anchored protein insertion cycle and the delivery of short secretory proteins to the translocon (Johnson et al., 2012
), either because the ER membrane receptor for Get3 is absent or because cellular energy levels are significantly depleted.
Our characterization of Get3 localization under glucose starvation substantially extends the previous observation that Get3 and tail-anchored protein substrates are found in aggregates in cells lacking the GET receptor (Schuldiner et al., 2008
). First, the kinetics and reversibility of the process () and its dependence on other cytosolic GET components () argue against an uncontrolled aggregation process. Second, even in the absence of the GET receptor, Get3 remains sensitive to glucose starvation, which maximizes its colocalization with a model tail-anchored client (). This glucose sensitivity in subcellular localization is intimately connected to Get3’s ability to hydrolyze ATP () suggesting that Get3 may directly respond to cellular energy status. In support of this hypothesis, we have excluded a major glucose-sensing signal transduction cascade that involves the yeast AMP-activated kinase Snf1/4 as a putative pathway responsible for the glucose-dependent relocalization of Get3 ().
Cellular quality control is aimed at preventing protein aggregation. However, it becomes increasingly clear that cells possess a second line of defense once the factors involved in counteracting aggregation are overwhelmed (Tyedmers et al., 2010
). Live-cell imaging has been key to obtaining insight into the cellular strategies by which aggregating proteins are directed to specific deposition sites and hence spatially segregated from processes that they might interfere with (Kaganovich et al., 2008
). Subsequent work has shown that different molecular chaperones are targeted to different types of deposition sites in a complex and dynamic pattern (Specht et al., 2011
). Two complementary methods of immuno-electron microscopy unequivocally demonstrate the presence of Get3-GFP at protein-dense foci with fibrillar appearance (). The cytosolic GET complex comprising Sgt2, Get4 and Get5, is implicated in transferring nascent tail-anchored protein precursors from the ribosome to Get3. Because the details of this transfer and loading process are poorly understood it was critical to determine whether ribosomes are present in the Get3-GFP-positive foci observed under glucose starvation. High-pressure freezing allows the detection of ribosomes by electron microscopy and in combination with immunolabeling Get3-GFP, we were able to reveal that ribosomes are absent from Get3-GFP-positive sites (). This result suggests that the cytosolic GET complex is more than a ribosome-associated transfer complex and exerts control over Get3-client complexes even after loading.
At the deposition sites, Get3 strikingly colocalizes with molecular chaperones that can prevent the aggregation of proteins exposing hydrophobic regions and/or reactivate aggregated proteins (). The dual function of Get3 as a holdase chaperone and a targeting factor for post-translational membrane integration may enable fast adaptation of the cell once cellular energy status has improved: Precursor proteins can remain associated with Get3 unless they require the activity of another factor present at the deposition site. The organization of various types of chaperones into larger assemblies controlling aggregation-prone proteins implies that clients can be transferred between effector proteins that decide and execute their fate. The problem of sorting hydrophobic clients between different chaperones that recognize precisely this quality – albeit with very different outcomes for the client protein – has previously been recognized and addressed by elegant in vitro
assays (Wang et al., 2010
; Hegde and Keenan, 2011
). However, our study is the first to define the physiological conditions and cellular structures that integrate the capacity of the GET pathway for hydrophobic protein sorting into global cellular proteostasis. Furthermore, our finding that Get3 acts as a holdase chaperone for clients other than tail-anchored proteins () raises the possibility that this activity can protect other classes of aggregation-prone proteins. Our findings also question the notion that membrane integration is the only fate available to a client protein once it has been sorted into a complex with Get3 (). A broader contribution of Get3 to cellular quality control is strongly supported by the intricate functional relationship between Get3 and the ubiquitin-proteasome system (Auld et al., 2006
) and the wider interaction networks formed by GET associated factors such as Sgt2 in yeast and SGTA and Bag6 in mammalian cells (Chang et al., 2010
; Leznicki et al., 2010
; Mariappan et al., 2010
; Wang et al., 2010
; Chartron et al., 2011
; Hegde and Keenan, 2011
; Hessa et al., 2011
; Kohl et al., 2011
; Chartron et al., 2012
; Leznicki and High, 2012
We have used two model clients, citrate synthase and luciferase, to monitor Get3’s ability to prevent protein aggregation upon chemical or thermal denaturation (). Both proteins contain stretches of ca. 20 amino acids that are rich in hydrophobic residues (http://dgpred.cbr.su.se/
; Hessa et al., 2007
). Whilst these hydrophobic regions will normally be buried in the hydrophobic core of the enzymes and interruptions by polar residues clearly distinguish them from transmembrane segments they will be exposed upon denaturation and might be recognized by Get3. Interestingly, maximal protection of either client from aggregation required at least a fourfold molar excess of Get3 consistent with the idea that higher-order complexes of Get3 are involved in the holdase function. The recent demonstration that a tetramer of an archeal Get3 homologue forms a hydrophobic chamber capable of accommodating a transmembrane segment (Suloway et al., 2011
) raises the possibility that the observed holdase activity involves the occlusion of aggregation-prone hydrophobic surfaces in a cage structure formed by a Get3 tetramer. The observed inhibitory effect of adenine nucleotides on the holdase activity () is compatible with the intricate structural interplay of the nucleotide-binding domain and the helices involved in forming the hydrophobic binding site of Get3 (Hegde and Keenan, 2011
; Chartron et al., 2012
). Furthermore, the negative impact of adenine nucleotides on Get3 holdase activity provides an important link to our observations regarding the localization of Get3 in vivo
: Get3 changes its localization when cells are glucose-starved or when alteration of a critical residue by sited-directed mutagenesis interferes with its ATPase activity (). Whilst the tail-anchored protein insertion cycle requires ATP hydrolysis, Get3 will be in a position to exert its holdase function under conditions of energy depletion when hydrophobic proteins accumulate.
It has not been rigorously determined how the threat to cellular proteostasis from different classes of aggregation-prone proteins adds up. The heat sensitivity of the get
mutants (Shen et al., 2003
; Metz et al., 2006
; Schuldiner et al., 2008
) may be explained by the accumulation of membrane protein precursors that jeopardize the capacity of the cell to withstand the load of misfolded proteins caused by heat shock. Our results clarify this point by clearly demonstrating that the chaperone Hsp104-GFP senses the presence of cytosolically accumulating tail-anchored protein precursors () and that its localization with respect to clients and Get3 depends on the precise nature of the proteotoxic stress. In conclusion we show that the capacity of Get3 to shield hydrophobic proteins is integrated into a spatially controlled and intricately regulated network of activities that probe, retain, reactivate or degrade hydrophobic clients. The possibilities for sorting clients between these individual activities are only beginning to emerge.