Raymond J. Deshaies

The role of stress proteins in membrane biogenesis

Abstract Stress proteins are synthesized by eukaryotic cells in response to diverse environmental insults. The properties of the 70 kDa and 90 kDa members of the heat shock and glucose-regulated protein families have been studied intensively, though their functions have proved difficult to determine. Recent work has revealed that several constitutively expressed homologues of these proteins participate in the assembly of biological membranes.

Structural and functional dissection of Sec62p, a membrane-bound component of the yeast endoplasmic reticulum protein import machinery.

SEC62 is required for the import of secretory protein precursors into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae. The DNA sequence of SEC62 predicts a 32-kDa polypeptide with two potential membrane-spanning segments. Two antisera directed against different portions of the SEC62 coding region specifically detected a 30-kDa polypeptide in cell extracts. A combination of subcellular fractionation, detergent and alkali extraction, and indirect immunofluorescence studies indicated that Sec62p is intimately associated with the ER membrane. Protease digestion of intact microsomes and analysis of the oligosaccharide content of a set of Sec62p-invertase hybrid proteins suggested that Sec62p spans the ER membrane twice, displaying hydrophilic amino- and carboxy-terminal domains towards the cytosol. Sec62p-invertase hybrid proteins that lack the Sec62p C terminus failed to complement the sec62-l mutation and dramatically inhibited the growth of sec62-l cells at a normally permissive temperature. The inhibitory action of toxic Sec62p-invertase hybrids was partially counteracted by the overexpression of Sec63p. Taken together, these data suggest that the C-terminal domain of Sec62p performs an essential function and that the N-terminal domain associates with other components of the translocation machinery, including Sec63p.

Dbf2–Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis

Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2–Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb–copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram’s tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.

Genetic dissection of the early stages of protein secretion in yeast

The earliest events in the export of secretory proteins from eukaryotic cells are their insertion into and transport across the membrane of the endoplasmic reticulum, followed by signal peptide cleavage and transfer of core oligosaccharides to specific asparagine residues. Much has been learned through reconstitution of these processes in vitro using cell-free extracts prepared from mammalian and yeast cells. Now, a combination of genetic, molecular and biochemical approaches are being employed to study the early stages of protein secretion in the yeast Saccharomyces cerevisiae.

Drug discovery: Fresh target for cancer therapy.

The Byzantine system for degrading proteins inside cells is already the target of a successful anticancer drug. A compound that inhibits another part of this system also shows promise in models of cancer in mice.

Structural biology: Corralling a protein-degradation regulator

The crystal structure of the COP9 signalosome, a large protein complex that regulates intracellular protein degradation, reveals how the complex achieves exquisite specificity for its substrates.