Bombing My Mind

A paper from Itoh et al. has stuck with me since its publication date 8 years ago for many reasons, but primarily because it stretched my mind and pleased my eye.The major conclusion of the paper—that Mind bomb-mediated ubiquitination of Delta promotes its endocytosis, thereby activating Notch signaling in an adjacent cell—was an important advance in the fields of ubiquitin-mediated regulation, Delta-Notch activated signaling, and neuronal development. For these reasons the title and abstract caught my attention. However, this manuscript was a challenging read for a life-long yeast cell biologist. I had heard talks over the years from my developmental biology colleagues, but I wasn't used to looking carefully at zebrafish embryos. I had also never rigorously thought through the experiments that would be needed to test whether a signal was acting cell autonomously in vivo. The paper therefore took time, but it was well worth it: the data are spectacular, both in clarity and aesthetics, and the conclusions are significant. A paper with a protein named Mind bomb, magenta and green fluorescent micrographs, and a description of a new ubiquitin ligase has everything it takes to make my day.This PaperPick relates to “Mind Bomb Is a Ubiquitin Ligase that Is Essential for Efficient Activation of Notch Signaling by Delta” by M. Itoh, C.-H. Kim, G. Palardy, T. Oda, Y.-J. Jiang, D. Maust, S.-Y. Yeo, K. Lorick, G.J. Wright, L. Ariza-McNaughton, A.M. Weissman, J. Lewis, S.C. Chandrasekharappa, and A.B. Chitnis, published in January 2003.Video AbstractThe first author of the original Developmental Cell paper, Dr. Motoyuki Itoh, introduces the mind bomb mutation, the work he did on it in Dr. Ajay Chitnis's laboratory, and some unanswered questions surrounding Notch ligand ubiquitination that persist to this day

The conserved Pkh–Ypk kinase cascade is required for endocytosis in yeast

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast α-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base–regulated serine–threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum– and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in α-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in α-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery

Solution Structure of a CUE-Ubiquitin Complex Reveals a Conserved Mode of Ubiquitin Binding

AbstractMonoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins

Calmodulin Promotes N-BAR Domain-Mediated Membrane Constriction and Endocytosis

Membrane remodeling by BAR (Bin, Amphiphysin, RVS) domain-containing proteins, such as endophilins and amphiphysins, is integral to the process of endocytosis. However, little is known about the regulation of endocytic BAR domain activity. We have identified an interaction between the yeast Rvs167 N-BAR domain and calmodulin. Calmodulin-binding mutants of Rvs167 exhibited defects in endocytic vesicle release. In vitro, calmodulin enhanced membrane tubulation and constriction by wild-type Rvs167 but not calmodulin-binding-defective mutants. A subset of mammalian N-BAR domains bound calmodulin, and co-expression of calmodulin with endophilin A2 potentiated tubulation in vivo. These studies reveal a conserved role for calmodulin in regulating the intrinsic membrane-sculpting activity of endocytic N-BAR domains