Sebastian Schuck

ESCRT proteins remodel membranes at many cell organelles, including the endoplasmic reticulum (ER). Here, we investigate whether ESCRTs in budding yeast participate in stress-induced ER reorganisation. We find that ER stress triggers the formation of tubular ER subdomains that recruit various ESCRT proteins. Recruitment of the major ESCRT-III protein Snf7 is mediated by the ESCRT-associated protein Bro1, a homologue of human ALIX, in a manner that is mechanistically distinct from Bro1 function at endosomes. ESCRT-containing ER subdomains are derived from ceramide-rich ER exit sites and form contacts with the Golgi. Furthermore, Bro1 helps to concentrate the tethering and lipid transfer protein Tcb3, a homologue of human extended synaptotagmins, at these organelle contacts and contributes to cellular fitness when lipid metabolism is perturbed. These results indicate that specialised ER exit sites can be repurposed for contacting the Golgi directly and uncover ESCRTs as organisers of stress-inducible ER-Golgi contacts that help maintain cell homeostasis.

The auxin system for inducible protein degradation is a powerful tool to investigate protein function. It consists of a degron fused to a target protein, an auxin-related ligand that binds to the degron, and a receptor that recognizes the auxin-bound degron and mediates proteasomal degradation of the target protein. Variants of all system components are available, and we here test three degrons, three auxins and three degron receptors to identify optimal combinations of these variants in budding yeast. We show that the degrons mIAA7 or AID* together with adamantyl-auxin and the degron receptor OsTIR1(F74G) allow particularly rapid and extensive degradation. Basal degradation in the absence of auxin is generally low and can be minimized further by inducible expression of OsTIR1(F74G). Moreover, we demonstrate that the remarkable efficiency of this system makes it competitive with established chemical inhibitors, such as tunicamycin and MG132, and with temperature-sensitive conditional alleles. These findings will aid the effective application of the auxin system.

Sophisticated techniques are available for systematic studies of yeast cell biology. However, it remains challenging to investigate protein subcellular localization changes on a proteome-wide scale. Here, we apply Dynamic Organellar Mapping by label-free mass spectrometry to detect localization changes of native, untagged proteins during endoplasmic reticulum (ER) stress. We find that hundreds of proteins shift between cellular compartments. For example, we show that numerous secretory pathway proteins accumulate in the ER, thus defining the extent and selectivity of ER retention of misfolded proteins. Furthermore, we identify candidate cargo proteins of the ER reflux pathway, determine constituents of reticulon clusters that segregate from the remainder of the ER and provide evidence for altered nuclear pore complex composition and nuclear import. These findings uncover protein relocalization as a major aspect of cellular reorganization during ER stress and establish Dynamic Organellar Maps as a powerful discovery tool in yeast.

The auxin system for inducible protein degradation is a powerful tool to investigate protein function. It consists of a degron fused to a target protein, an auxin-related ligand that binds to the degron, and a receptor that recognises the auxin-bound degron and mediates proteasomal degradation of the target protein. Variants of all system components are available, and we here test three degrons, three auxins and three degron receptors to identify optimal combinations of these variants in budding yeast. We show that the degrons mIAA7 or AID* together with adamantyl-auxin and the degron receptor OsTIR1(F74G) allow particularly rapid and extensive degradation. Basal degradation in the absence of auxin is generally low and can be prevented entirely by inducible expression of OsTIR1(F74G). Finally, we demonstrate that the remarkable efficiency of this system makes it competitive with established chemical inhibitors, such as tunicamycin and MG132, and with temperature-sensitive mutant alleles. These findings will aid the effective application of the auxin system.

Sophisticated techniques are available for systematic studies of yeast cell biology. However, it remains challenging to investigate protein subcellular localization changes on a proteome-wide scale. Here, we apply Dynamic Organellar Maps (DOMs) by label-free mass spectrometry to detect localization changes of native, untagged proteins during endoplasmic reticulum (ER) stress. We find that hundreds of proteins shift between cellular compartments. For example, we show that numerous secretory pathway proteins accumulate in the ER, thus defining the extent and selectivity of ER retention of misfolded proteins. Furthermore, we identify candidate cargo proteins of the ER reflux pathway, determine constituents of reticulon clusters that segregate from the remainder of the ER and provide evidence for altered nuclear pore complex composition and nuclear import. These findings uncover protein relocalization as a major aspect of cellular reorganization during ER stress and establish DOMs as a powerful discovery tool in yeast.

One way cells control the speed and specificity of protein degradation is by regulating the activity of ubiquitin ligases. Upon proteotoxic stress in yeast, the intrinsically disordered protein Roq1 binds the ubiquitin ligase Ubr1 as a pseudosubstrate, thereby modulating the degradation of substrates of the N-degron pathway and promoting the elimination of misfolded proteins. The mechanism underlying this reprograming of Ubr1 is unknown. Here, we show that Roq1 controls Ubr1 by means of two cooperating multifunctional motifs. The N-terminal arginine and a short hydrophobic motif of Roq1 interact with Ubr1 as part of a heterobivalent binding mechanism. Via its N-terminal arginine, Roq1 regulates the ubiquitination of various N-degron substrates and folded proteins. Via its hydrophobic motif, Roq1 accelerates the ubiquitination of misfolded proteins. These findings reveal how a small, intrinsically disordered protein with a simple architecture engages parallel channels of communication to reprogram a functionally complex ubiquitin ligase.

One way cells control the speed and specificity of protein degradation is by regulating the activity of ubiquitin ligases. Upon proteotoxic stress in yeast, the intrinsically disordered protein Roq1 binds the ubiquitin ligase Ubr1 as a pseudosubstrate, thereby modulating the degradation of substrates of the N-degron pathway and promoting the elimination of misfolded proteins. The mechanism underlying this reprograming of Ubr1 is unknown. Here, we show that Roq1 controls Ubr1 by means of two cooperating multifunctional motifs. The N-terminal arginine and a short hydrophobic motif of Roq1 interact with Ubr1 as part of a heterobivalent binding mechanism. Via its N-terminal arginine, Roq1 regulates the ubiquitination of various N-degron substrates and folded proteins. Via its hydrophobic motif, Roq1 accelerates the ubiquitination of misfolded proteins. These findings reveal how a small, intrinsically disordered protein with a simple architecture engages parallel channels of communication to reprogram a functionally complex ubiquitin ligase.

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Autophagy is fundamental for cell and organismal health. Two types of autophagy are conserved in eukaryotes: macroautophagy and microautophagy. During macroautophagy, autophagosomes deliver cytoplasmic constituents to endosomes or lysosomes, whereas during microautophagy lytic organelles take up cytoplasm directly. While macroautophagy has been investigated extensively, microautophagy has received much less attention. Nonetheless, it has become clear that microautophagy has a broad range of functions in biosynthetic transport, metabolic adaptation, organelle remodeling and quality control. This Review discusses the selective and non-selective microautophagic processes known in yeast, plants and animals. Based on the molecular mechanisms for the uptake of microautophagic cargo into lytic organelles, I propose to distinguish between fission-type microautophagy, which depends on ESCRT proteins, and fusion-type microautophagy, which requires the core autophagy machinery and SNARE proteins. Many questions remain to be explored, but the functional versatility and mechanistic diversity of microautophagy are beginning to emerge.