The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4 -green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4 -green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeastSaccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In thedoa4Δ mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Δ cells. This loss of viability and several other defects of doa4Δ cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.

Degradation of many eukaryotic proteins requires their prior ligation to polyubiquitin chains, which target substrates to the 26S proteasome, an abundant cellular protease. We describe a yeast deubiquitinating enzyme, Ubp14, that specifically disassembles unanchored (‘free’) ubiquitin chains in vitro, a specificity shared by mammalian isopeptidase T. Correspondingly, deletion of the UBP14 gene from yeast cells results in a striking accumulation of free ubiquitin chains, which correlates with defects in ubiquitin‐dependent proteolysis. Increasing the steady‐state levels of ubiquitin chains in wild‐type cells (by expressing a derivative of ubiquitin with an altered C‐terminus) inhibits protein degradation to a degree comparable with that observed in ubp14Δ cells. Inhibition of degradation is also seen when an active site mutant of Ubp14 is overproduced in vivo. Surprisingly, overproduction of wild‐type Ubp14 can inhibit degradation of some proteins as well. Finally, Ubp14 and human isopeptidase T are shown to be functional homologs by complementation analysis. We propose that Ubp14 and isopeptidase T facilitate proteolysis in vivo by preventing unanchored ubiquitin chains from competitively inhibiting polyubiquitin–substrate binding to the 26S proteasome.

In keeping with the growing movement in scientific publishing toward transparency in data and methods, we propose changes to journal authorship policies and procedures to provide insight into which author is responsible for which contributions, better assurance that the list is complete, and clearly articulated standards to justify earning authorship credit. To accomplish these goals, we recommend that journals adopt common and transparent standards for authorship, outline responsibilities for corresponding authors, adopt the Contributor Roles Taxonomy (CRediT) (docs.casrai.org/CRediT) methodology for attributing contributions, include this information in article metadata, and require authors to use the ORCID persistent digital identifier (https://orcid.org). Additionally, we recommend that universities and research institutions articulate expectations about author roles and responsibilities to provide a point of common understanding for discussion of authorship across research teams. Furthermore, we propose that funding agencies adopt the ORCID identifier and accept the CRediT taxonomy. We encourage scientific societies to further authorship transparency by signing on to these recommendations and promoting them through their meetings and publications programs.

Egr-1 is an immediate-early response gene induced transiently and ubiquitously by mitogenic stimuli and also regulated in response to signals that initiate differentiation. The Egr-1 gene product, a nuclear phosphoprotein with three zinc fingers of the Cys2His2 class, binds to the sequence CGCCCCCGC and transactivates a synthetic promoter construct 10-fold in transient-transfection assays. We have analyzed the structure and function of the Egr-1 protein in detail, delineating independent and modular activation, repression, these results and points to a role for sustained Egr-1 expression in these differentiated cell types. In the developing mouse, the predominant expression of Egr-1 in cartilage and bone, coordinate with c-fos induction, suggests a role for these coregulated genes in skeletal development (68). Recently, Nguyen et al. have shown that Egr-1 is essential for macrophage differentiation of the myeloid leukemia cell line HL60 and further that Egr-1 functions to restrict differentiation, since constitutive Egr-1 expression is incompatible with the differentiation of HL60 cells along the granulocyte lineage (50). Characterization of the Egr-1 gene product has shown that this serine/threonine/proline-rich nuclear phosphoprotein (6,12,73) activates transcription through the sequence CGC CCCCGC in transient transfection assays (6,8,36,53). Recently, Pavletich and Pabo have cocrystallized the three zinc fingers of Egr-1 with its cognate sequence, defining a DNA-binding domain (54). However, to date the regions responsible for activation and nuclear localization have not been mapped. A body of work suggests the modular nature of transcription factors, in which functional domains are structurally independent and able to confer activity on heterologous proteins (reviewed in reference 55). We have used deletion analysis and gene fusions to dissect the functional domains of Egr-1. We report here that the serine/ threonine-rich N terminus of Egr-1 encodes its transactivation function. Remarkably, a compact 34-amino-acid region, residues 281 to 314, represses transcription in gene fusion experiments. The zinc finger domain encodes the DNAbinding activity, and this region in conjunction with a basic sequence 5' of the fingers is responsible for nuclear localization of Egr-1. 4556 on May 11, 2018 by guest