The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo.

Abstract

The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally shortlived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MATa2 repressor of the yeast Saccharemyces cerevisiae is an extremely short-lived protein. We now demonstrate that a2 is conjugated to ubiquitin in vivo. More than one lysine residue of a2 can bejoined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain.Overexpression of degradation-impaired ubiquitin variants was used to show that at least a ficant fraction of a2 degradation is dependent on its ubiquitination.The temporal control of many cellular processes involves short-lived regulators. Proteins that are either conditionally or constitutively short-lived in vivo include cell cycle regulators such as the cyclins (1) and proteins controlling cell differentiation and embryonic development (2-5).A distinct class of intracellular proteolytic pathways involves the covalent ligation of a 76-residue protein, ubiquitin (Ub), to the E-amino groups of acceptor proteins, which are thereby marked for degradation (6). Ub-protein conjugation is catalyzed by a family of Ub-conjugating enzymes, also called E2 enzymes (7). For many short-lived intracellular proteins, their conjugation to Ub is apparently essential for their degradation (8, 9). At the same time, the only specific examples of naturally short-lived proteins whose degradation appears to involve ubiquitination are the phytochrome photoreceptor of plants (10) and cyclins, a family of cell cycle regulators (11).Our recent studies of short-lived proteins included the cell type-specific transcriptional repressor MATa2 of the yeast Saccharomyces cerevisiae (3). This eukaryote has two haploid cell types, a and a, which can mate to form an a/a diploid (12). Ultimately, cell identity is determined by the matingtype, or MAT, locus. In homothallic S. cerevisiae strains, unexpressed a or a information at two other genomic sites is copied into the MAT locus during a mating-type switch.Interconversion between a and a cell types can occur in <90 min. Selective protein degradation might underlie the matingtype switch by causing rapid disappearance of the regulatory proteins involved once their synthesis has ceased.Indeed, the a2 repressor, encoded by the MATa locus and involved in the repression of a cell-specific genes in a cells and of haploid cell-specific genes in a/a diploids, has an in vivo half-life of only -5 min at 30°C (3). Both the aminoterminal and the carboxyl-terminal domains of a2 contain regions that can act as autonomous degradation signals; these signals operate via genetically distinguishable pathways (3).We now demonstrate, using epitope-tagged forms of ubiquitin, that a2 is ubiquitinated in vivo. We also show that ubiquitination of a2 contributes to its rapid degradation.

Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2

“…variety of in vivo substrates of the ubiquitin conjugating system have been identified, including histones [10], actin [11], cytlins [12], the tumor suppressor p53 [13,14], MATch2 transcr ption repressor [15], cell surface receptors [16][17][18], c-jun [19] and NF~cB [20]. Some ubiquitin conjugating enzymes are able to transfer ubiquitin to the model substrate histone in vitro without any accessory factors, such as E3s.…”

The Transmembrane Domain of Acid Trehalase Mediates Ubiquitin-independent Multivesicular Body Pathway Sorting

“…To verify this result and further test whether ubiquitination occurs on the lysine residues of the Ath1 cytosolic tail, we carried out immunoprecipitation experiments in cells expressing ubiquitin tagged with HA (HA-Ub; Hochstrasser et al, 1991). We used the doa4⌬ background to reduce deubiquitination and potentially increase the pool of ubiquitinated Ath1.…”

“…The plasmid YEp112 (pHA-Ub; Hochstrasser et al, 1991) was generously provided by Dr. Mark Hochstrasser (Yale University).…”

“…The corresponding gene products are referred to as GFP-Ath1polarmut and GFP-Ath1⌬Npolarmut. DNA sequencing was used to verify all of the introduced point mutations.The plasmid YEp112 (pHA-Ub; Hochstrasser et al, 1991) was generously provided by Dr. Mark Hochstrasser (Yale University). …”

Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the doa4 Mutant

“…It is possible that this minor fraction has a different intracellular distribution than the bulk of Ste6. To be able to detect ubiquitinated Ste6, the plasmid YEp112 (Hochstrasser et al, 1991) carrying a hemeagglutin (HA)-tagged ubiquitin variant, under the control of the copper-inducible CUP1 promoter, was cotransformed into the ⌬ste6 strain RKY959 together with a multicopy STE6 plasmid. Ste6 overproduced from a multicopy plasmid showed the same half-life and distribution on sucrose gradients as Ste6 expressed form the chromosomal copy of the STE6 gene.…”