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1.  Substrate binding on the APC/C occurs between the co-activator CDH1 and the processivity factor DOC1 
The anaphase–promoting complex/cyclosome (APC/C) is a 22 S ubiquitin ligase complex that initiates chromosome segregation and mitotic exit. We have used biochemical and electron microscopic analyses of Saccharomyces cerevisiae and human APC/C to address how the APC/C subunit Doc1 contributes to recruitment and processive ubiquitylation of APC/C substrates, and to understand how APC/C monomers interact to form a 36 S dimeric form. We show that Doc1 interacts with Cdc27, Cdc16 and Apc1, and is located in vicinity of the cullin–RING module Apc2–Apc11 in the inner cavity of the APC/C. Substrate proteins also bind in the inner cavity, in close proximity to DOC1 and the co–activator CDH1, and induce conformational changes in APC2–APC11. Our results suggest that substrates are recruited to the APC/C by binding to a bipartite substrate receptor composed of a co–activator protein and Doc1.
PMCID: PMC4300845  PMID: 21186364
2.  Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase 
Molecular Cell  2014;53(3):471-483.
Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.
Graphical Abstract
•The Atg1 kinase phosphorylation consensus was identified on peptide arrays•Atg9 is a direct target of the Atg1/ULK1 kinase in vitro and in vivo•Atg9 phosphorylation recruits Atg18 and Atg8 to the PAS•Atg9 phosphorylation is required for isolation membrane expansion/autophagy function
Autophagy function is pivotal to cell health. Papinski et al. identify the phosphorylation consensus of the central kinase in this pathway, Atg1. The autophagy-related protein Atg9 is a direct target of Atg1. Atg9 phosphorylation by Atg1 is required for autophagosome formation. This finding sheds light on how Atg1 controls autophagy.
PMCID: PMC3978657  PMID: 24440502
4.  Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation 
The EMBO journal  2012;31(22):4304-4317.
Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5-Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the preautophagosomal structure but is essential for autophagy and cytoplasm-to-vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5-Atg12/Atg16 complex during autophagosome formation.
PMCID: PMC3501226  PMID: 23064152
autophagy; autophagosome; Atg5; Atg8; Atg16
5.  Reticulophagy and Ribophagy: Regulated Degradation of Protein Production Factories 
During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.
PMCID: PMC3299282  PMID: 22481944
6.  Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress 
The EMBO Journal  2011;30(16):3274-3284.
Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress
Here, the Hog1 kinase interacts with and activates the ubiquitin protease Ubp3 in a stress-dependent manner. The phosphorylation of Ubp3 enhances RNA polymerase II occupancy on osmotic stress-responsive genes.
Protein ubiquitylation is a key process in the regulation of many cellular processes. The balance between the activity of ubiquitin ligases and that of proteases controls the level of ubiquitylation. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK has a key role in reprogramming the gene expression pattern required for cell survival upon osmostress. Here, we show that the Ubp3 ubiquitin protease is a target for the Hog1 SAPK to modulate gene expression. ubp3 mutant cells are defective in expression of osmoresponsive genes. Hog1 interacts with and phosphorylates Ubp3 at serine 695, which is essential to determine the extent of transcriptional activation in response to osmostress. Furthermore, Ubp3 is recruited to osmoresponsive genes to modulate transcriptional initiation as well as elongation. Therefore, Ubp3 activity responds to external stimuli and is required for transcriptional activation upon osmostress.
PMCID: PMC3160652  PMID: 21743437
gene expression; Hog1; osmostress; SAPK; Ubp3
7.  Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast 
Science signaling  2010;3(153):rs4.
The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules, and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery, and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.
PMCID: PMC3072779  PMID: 21177495

Results 1-7 (7)