PMC

Distribution of K0 Ub with ubiquitin chain-linkages. A, The ratios of the main Ub linkages, Lys48 and Lys63 in whole cell extract (K0/WT Ub) were determined by SILAC according to MW regions. For MW regions <130 kDa, Orbitrap detection of signature peptides (supplemental Fig. S6) was performed with a mass inclusion list. B, Ratio of total conjugated Ub in cells expressing K0 Ub relative to cells expressing WT Ub based on the MS intensity ratio of the tryptic peptide EGIPPDQQR (see explanation in supplemental Fig. S6). C, Penetration of K0 Ub into the ubiquitin landscape according to MW. Ubiquitin in the SILAC cells comes from two sources: endogenous (WT) and expressed tagged ubiquitin (either WT or K0). The ratio of extendable Ub (derived from peptide TITLEVESSDTIDNVK) to total ubiquitin (derived from peptide EGIPPDQQR) between strains provides a measure of K0 Ub penetration (details in supplemental Fig. S6). D, Main ubiquitin linkages pulled out with tagged ubiquitin. Bars reflect ratio of linkages associated with tagged-K0 Ub relative to tagged-WT Ub in each MW region. K0 Ub is preferentially associated with Lys63 linkages over Lys48 in affinity purified chains.

Lysineless (K0) Ub penetrates into the conjugated Ub pool increasing mono/poly Ub ratio. A, WT yeast cells were transformed with plasmids for expression of either RGS-His₈ tagged lysineless Ub (K0 Ub) or with tagged WT Ub as control. Rapidly lysed whole cell extracts were resolved by 18% SDS-PAGE, transferred and immunoblotted with anti RGS-His antibodies (left panel) or with anti Ub antibodies (right panel). B–D, Equal amounts of cells expressing WT Ub, K0 Ub, or empty vector (serving as background strain) were lysed and whole-cell extracts resolved by gradient SDS-PAGE. The high MW region was excised and subjected to Ub-AQUA analysis as described in . B, As in , expression of WT Ub from a plasmid did not significantly alter total conjugates relative to background strain (empty vector). Ubiquitin levels in both strains were normalized using common internal peptide standards. K0 Ub expression resulted in a roughly 50% increase in total ubiquitin conjugates. C, Increase in mono/end cap modifications accounts for bulk of ubiquitin conjugates accumulating in K0 Ub expressing cells relative to nontransformed cells. D, The relative abundance of different Ub modifications in cells expressing K0 Ub is displayed as a percentage of total cellular conjugated Ub.

Impact of K0 Ub expression on proteome and ubiquitinome. A, Schematic description of SILAC approach used in this study. Cells expressing RGS-His₈ tagged WT or RGS-His₈ tagged K0 Ub were differentially isotopically labeled, mixed, lysed under denaturing conditions, and split for MW resolution of total proteome or for affinity purification of Ub-conjugates. MS/MS analysis was performed at three levels: proteins in total cell extract (proteome; panel B); proteins in pullout of ubiquitin-conjugates (ubiquitinome; panel C); and analysis of ubiquitin linkages (). B, 2196 different proteins were identified at high confidence in whole cell extracts of K0 Ub and WT Ub expressing cells. Enrichment factors were calculated from intensity of MS signals (log₂(K0/WT); supplemental Table S2). Bars represent frequency of proteins (%) in each enrichment factor range. The red dotted line represents a ratio of unity (log₂1 = 0); i.e. proteins whose ratio was unaltered by expression of K0 Ub. C, A total of 856 different proteins were identified by Ub-affinity pullout (supplemental Table S1). Enrichment factors were calculated and displayed as in panel B. D, The enrichment factor for each protein in the Ub pullout (y axis) was plotted against its enrichment factor in whole cell extract (x axis). The two dimensional plot indicates the K0 Ub-induced perturbation on ubiquitination status of each substrate relative to changes in its cellular abundance. A red asterisk represents the K0 Ub protein, which was added to the database of all possible yeast translated open reading frames. E, Biological pathways significantly enriched with ubiquitinated proteins upon K0 Ub expression. Identified proteins were classified into biological pathways using the AmiGO program. Ubiquitinated proteins with a greater than twofold increase in K0 Ub expressing cells were categorized according to biological pathways. The relative portion that each category makes up out of total ubiquitinated proteins is shown in red bars. Blue bars represent the portion that these pathways make up of known ubiquitinated targets (taken from supplemental Table S3). F, To emphasize the enrichment of ubiquitinated proteins in specific biological categories the span of enrichment factors for all proteins identified in the Ub pullout is displayed for each category (surrounding the average enrichment ratio marked as red squares). For comparison, the enrichment factors of these same proteins in whole cell extract are also shown along with the average enrichment in extract of proteins belonging to each category (green diamonds). The comparison highlights that proteins belonging to these categories accumulate as ubiquitinated conjugates in K0 Ub expressing cells.

The conjugated Ub linkage profile. A, A general scheme for determining the linkage profile of conjugated ubiquitin. Logarithmically growing WT yeast cells (genotype of this and other strains used in this study are listed in supplemental Table S7) were rapidly lysed and the resulting whole-cell extract was resolved by a 4–12% gradient SDS-PAGE. The high MW region, corresponding to the majority of the Ub conjugates (as verified by immunoblotting with anti-Ub polyclonal antibody; left panel), was excised and subjected to digestion by Trypsin (right panel). Isotope-labeled Ub AQUA peptides were added as internal standards, and mixture subjected to multiplexed liquid chromatography-selected reaction monitoring processing (Ub-AQUA peptide transitions are listed in supplemental Table S9). Ubiquitin-linkage determination was performed as described (, , , , ). B, TCA lysis reproducibly generates the highest yield of ubiquitin conjugates. Logarithmic yeast cells were processed according to scheme in panel A, differing only in the lysis protocol. Top: The total amount of conjugated ubiquitin detected under the different lysis conditions is shown in pmols. TCA lysis retained the highest amount of total ubiquitin in high MW conjugates compared with other lysis conditions. Middle: Quantification of Lys48 and Lys63 ubiquitin linkages captured under different lysis conditions. TCA lysis retained the highest absolute amounts of Lys48 linkages and significantly higher amounts of Lys63 chains than those trapped in a measurable form by other lysis conditions. Bottom: Quantification of mono and endcap ubiquitin under different lysis conditions. A large portion of ubiquitin found in high MW conjugates was not modified on any lysine residue. The highest portion of such ubiquitin representing mono and endcap modifications was obtained under TCA lysis, without detracting from amounts of polyUb chains trapped in same samples (middle panel). C, Summary of ubiquitin linkage distribution obtained under TCA lysis. The relative abundance of polyUb as well as nonextended ubiquitin (mono and end cap) displayed as a percentage of total conjugated ubiquitin. In this sample, chains linked via Lys6, or N terminus are scarce (<0.03%) and are therefore not shown in this chart. D, Lys48Arg (K48R) ubiquitin mutation alters normal partitioning between polyUb linkages. Equal amounts of WT cells transformed with plasmids overexpressing (oe) WT Ub or K48R Ub were processed as in panel A, and distribution of main ubiquitin modifications was compared with that in cells transformed with empty vector (serving as background strain). Properties of these and other plasmids used in this study are listed in supplemental Table S8. Overexpression of WT Ub did not significantly alter ubiquitin conjugates in high MW relative to background strain. Upon expression of K48R Ub, Lys48-linkages remained the same as in WT background, however as other linkages increased, the relative portion of Lys48-linkages decreased.

Induction of K0 Ub interferes with protein sorting more than it impacts ubiquitin-proteasome dependent degradation. A, Influence of K0 Ub induction on known ubiquitin-proteasome substrates. Cellular stability of ectopically expressed MyoD or Pcl5 in the presence of either WT or K0 Ub was monitored by addition of cycloheximide to exponentially growing yeast cells. Aliquots were taken at the indicated time points after addition of cycloheximide and analyzed by immunoblotting with anti-MyoD (top panel) or anti-Pcl5 (bottom) antibodies, and anti-PGK serving as loading control. B, Expression of K0 Ub increases the ubiquitination levels of Syp1. Cells expressing Syp1-GFP together with either RGS-His₈-K0 Ub or RGS-His₈-WT Ub were lysed and loaded onto a Ni-NTA column for isolation of ubiquitin conjugates. Syp1 content in whole cell extracts (WCE) and eluate of isolated ubiquitin conjugates (El) were analyzed by immunoblotting with anti-GFP antibodies. C, Expression of K0 Ub alters the ubiquitination pattern of Sna3. Cells expressing Sna3–6HA together with either WT or K0 Ub were lysed, immunoprecipitated with anti-HA antibodies, and analyzed for ubiquitinated Sna3 species. Immunoblotting the immunoprecipitate with anti-HA identified unmodified Sna3 as well as modified higher MW forms (left). Immunoblotting with anti His-tag confirmed the presence of K0 Ub in high MW conjugates of ubiquitinated Sna3 (right). D, Cells expressing K0 Ub display typical phenotypes of defected endocytosis: sensitivity to canavanine but resistance to nickel ions. Ten-fold serial dilutions of cells expressing either WT or K0 Ub were spotted onto selective medium (control) or media supplemented with either canavanine (1 μg/ml) or NiCl2 (1.5 mm), and grown at 30°. E, Expression of K0 Ub partially impairs MVB sorting of GFP-Phm5 and Sit1-GFP. Cells expressing GFP-Phm5 together with K0 or WT Ub were grown to midexponential phase in selective medium (carbon source glucose). Intracellular localization of GFP-Phm5 was examined by fluorescence microscopy. GFP-Phm5 localizes to vacuolar lumen in WT cells at steady state, whereas accumulation at vacuolar membrane periphery and endosomes occurs upon K0 Ub induction. Similar monitoring of Sit1 was performed after 1 h induction with galactose for the expression of Sit1-GFP. For this target too, K0 Ub interferes with vacuolar lumen sorting. F, Expression of K0 Ub partially impairs plasma membrane internalization and MVB sorting of the Jen1 transporter. Cells encoding for Jen1-GFP were transformed with either K0 or WT Ub. Cells were induced in lactic acid for 4 h for expression and plasma membrane targeting of Jen1-GFP. GFP fluorescence was monitored before (t = 0) and 30 min after (t = 30′) the addition of 2% glucose, which triggers endocytosis and vacuolar targeting of Jen1. Within 30 min, all GFP fluorescence was detected within vacololar lumen, whereas ∼10% of K0 Ub expressing cells retained vacuolar membrane and faint plasma membrane staining.