Carboxypeptidase Y (CPY) is a soluble vacuolar hydrolase that is sorted from the late Golgi to the late endosome and subsequently trafficked to the vacuole [12
]. In yeast, Sortin2 interfered with CPY sorting resulting in secretion to the media [1
]. Sortin2 also phenocopied vps
mutants without inhibiting yeast growth, at concentrations ranging from 4.7 μM to 47 μM (Fig and ). Upon Sortin2 treatment, there was no detectable presence in the medium of the proteins such as alkaline phosphatase (ALP), a membrane-bound vacuolar protein, or 3-phosphoglycerate kinase, a cytoplasmic protein (Fig. ). These controls indicated that CPY in the medium of Sortin2-treated cells was not due to cell lysis. The total amount of expressed CPY protein was not significantly affected by Sortin2 treatment indicating that its secretion was due to an alteration in cargo delivery rather than simple overloading of endomembrane compartments (Fig ). Therefore, we concluded that Sortin2 targeted components involved in the delivery of CPY from the endoplasmic reticulum to the vacuole.
Figure 1 Effects of Sortin2 treatments on S. cerevisiae. (A) Dot-blot secretion assay using monoclonal antibodies raised against CPY, alkaline phosphatase (ALP) and 3-phosphoglycerate kinase (PGK). (B) Yeast growth in presence of 1% DMSO (●), 2 μg/ml (more ...)
Sortin2 structure-bioactivity relationships
In order to understand the structural determinants of Sortin 2 necessary for bioactivity, a panel of chemicals that were similar in structure to Sortin2 were examined. The Sortin2 contains discrete domains: a chlorobenzene, a furan, a thiazolidine ring, and a sulphite group. The activity of structural analogs as promoters of CPY secretion is shown in Figure . For convenience, the potency of Sortin 2 and its analogues was defined as the minimum concentration at which activity was observed.
Structure-activity relationship of Sortin2 structural analogs. The ability to trigger secretion of CPY was tested for all compounds shown. Compounds are referred by the identification numbers assigned by manufacturer.
Chemical 1 (Fig. ) represented the Sortin2 structure minus the sulphite group. This analogue was inactive indicating that the sulphite group was critical for binding of Sortin2 to its target. Chemical 2 which had a benzoic ring substitution instead of the sulphite group had the same activity as Sortin2. Chemical 3 had a carboxyl group instead of the sulphite group. It was bioactive; however, it had a lower potency (40 μM) than Sortin2 and chemical 2 (5 μM). Taken together, these data suggest that the interaction between Sortin2 and its target probably required a dense electron cloud in order to affect target activity and function. This could be direct or indirect by affecting the overall conformation of Sortin2.
Chemical 4 lacked the chlorobenzene ring, but did not alter the delivery of CPY to the vacuole. We further tested the importance of the chlorine on the benzene ring by testing another chemical (5) which had a nitro group substitution. Chemical 5 was twenty times less active than Sortin2 which indicated that the presence of the chloride was important for full activity. However, chemical 6, in which the chloride was absent showed a potency that was similar to that of chemical 5 supporting the notion that the halogen atom was not a key feature for bioactivity. Chemical 7 which had two chlorides associated with the benzoic group was completely inactive, perhaps due to steric effects. Steric hindrance may also explain the low activity of chemical 5 since the major difference was the chloride and nitro groups. Overall we concluded that the sulphite group and the benzoic acid ring were essential for Sortin2 bioactivity.
Screening for yeast mutants hypersensitive to Sortin2
In order to identify cellular pathways affected by Sortin2 a haploid yeast deletion library was screened for mutants that secreted CPY at concentrations of Sortin2 that did not trigger detectable CPY secretion in the wild type haploid strain. Of the collection of 4,800 strains, 243 putative hypersensitive mutants were identified in the primary screen (Fig. ). Each putative mutant was subsequently exposed to various concentrations of Sortin2 to verify the drug dependency of their secretion phenotype (Fig. ). A mutant was considered verified as hypersensitive if significant CPY secretion was detected at a concentration of Sortin2 less than that necessary to result in CPY secretion from the parental line (Fig. ). Ninety percent (217) of the strains were verified as hypersensitive to Sortin 2. The large number of successfully verified strains demonstrated the robustness of the primary screen.
Figure 3 Identification of Sortin2 hypersensitive mutants. (A) Primary screen: 4827 haploid deletion strains were challenged with 1% DMSO (DMSO) or Sortin2 at 2 μg/ml (4.7 μM) (Sortin2). Culture media was analyzed for the presence of CPY by dot (more ...)
Vacuolar protein sorting (vps) mutants and hypersensitivity to Sortin2
Sixty-one yeast deletion strains were known previously to have impaired sorting of CPY; these are referred to as vps
mutants. Forty-one vps
mutants were identified as drug-dependent in our screen with respect to Sortin2. These included mutants in all six classes of VPS
genes (Additional file 1
). Class E is involved in protein sorting to lumenal vesicles of the MVB (multi-vesicular body). This family contains 16 members plus VTA1
; however, the latter two genes were not described as VPS
]. Remarkably, fourteen out of sixteen members (88.9%) of the VPS
Class E were hypersensitive to Sortin2, the class with highest representation among VPS
genes. Interestingly, the remaining 20 vps
mutants displayed no secretion of CPY when treated with Sortin2. The screen, thus, discriminated between deletion and Sortin2-induced vps
phenotypes. This indicated that the Sortin2 phenotype was specific and distinct, rather than a broad effect on secretion, when compared to the majority of the vps
mutants. As part of this screen, we analyzed the drug sensitivity of 148 strains identified previously in a screen for mutants that secreted CPY [15
]. Among these mutants, 91 were hypersensitive to Sortin2 when compared to the untreated control. The absence of a hypersensitive response from the other 57 mutants indicated that Sortin2 was selective for specific elements of the vacuole targeting machinery.
Analyses of identified ORFs
] was used to classify the Sortin2 hypersensitive dataset into functional categories relative to the Saccharomyces genome (Table , Additional file 2
). Some categories showed statistically significant over-representation such as "interaction with the environment" and "biogenesis of cellular components" which were enriched 1.7- and 1.5-fold, respectively (p ≤ 0.04) (Table ). The greatest enrichment were the categories "protein fate" and "cellular transport, transport facilities and transport routes" which displayed 2.5- and 3-fold enrichment, respectively (p = 1.51 E-19 and 2.99 E-29; Table ). In terms of subcellular location, the greatest over-representation corresponded to the category "endosomes" with a 17.5-fold enrichment compared to the yeast genome (p = 6.2 E-35) (Table ). The "Golgi apparatus", "transport vesicles", "vacuole" and "punctuate composite" categories were enriched between 3.5- and 5.4-fold with statistically significant over-representation as shown by the p-values in Table . These results revealed that processes within the endomembrane system are strongly and specifically affected by Sortin2 treatment.
Functional and locational gene product categorization of Sortin2 hypersensitive ORFs.
Forty-seven percent of the Sortin2 dataset was associated with the GO term "Transport". The set was analyzed by Term Finder tools, and significantly, the sole descendant terms included "Protein Transport", "Intracellular Transport", "Vesicle-mediated Transport" and "Secretory Pathway" highlighting the involvement of the majority of identified ORFs in protein trafficking pathways (data no shown). We also applied the Term Finder tool to analyze the association of the Sortin2 dataset with granular GO terms that were linked to a cellular component (Additional file 3
). Within the Sortin2 hypersensitive dataset, we identified members of crucial Golgi and endosomal sorting complexes, their interacting partners and other resident proteins associated all directly in CPY sorting (Additional file 3
). In the endosomal compartment, which is the destination for CPY after exiting the late Golgi, we identified members of the three known endosomal and the MVB sorting complexes. Systematic deletion of ESCRT (Endosomal Sorting Complex Required for Transport) genes was reported to cause reduced telomere length [17
]. Interestingly, we identified ESCRT-unrelated telomere length mutants as hypersensitive in our screen. In addition several mutants linked to chromatin remodelling were identified (Additional file 3
). Overall, we concluded that the Sortin2 hypersensitive ORFs were located preferentially within the endomembrane system and included many molecular components related to the CPY delivery. Our screening results also supported the hypothesis previously proposed that interactivity occurs between the endomembrane system, chromatin remodelling, and telomere maintenance processes.
Importantly, the sublethal conditions of the screening assay yielded an unbiased set of mutants. The dataset did not include any ABC transporters or ergosterol biosynthetic genes which are related to multi-drug resistance [18
] suggesting that the screen distinguished between induced vps
phenotypes and general drug sensitivity.
Of 217 Sortin2 hypersensitive mutants, 30 were classified as unknown according to the SGD databases. Among these ORFs, several were dubious genes with known genes on the complementary DNA strand. It is also possible that the Sortin2 phenotype resulted from a defect in an authentic gene whose ORF overlapped the dubious ORF. These dubious genes are listed with their corresponding known ORFs in parenthesis in Table . YIP4
encodes a protein that mediates vesicular trafficking via its interaction with Rab GTPases [19
] supporting the dubious character of YGL199C
. Similarly, Gim5p is a member of a protein complex that promotes formation of functional α- and γ-tubulin [20
]. This complex includes Gim4p that was identified in our screen; thus YML094C-A
is probably a dubious ORF.
Unknown function ORF of the corresponding deletion strains identified as Sortin2 hypersensitive mutants.
Other unknown genes were found in high-throughput screens that focused on genetic/physical interactions and protein localization [21
]. This annotation supports putative roles for the corresponding gene products in protein trafficking. Ygr206wp, for example, was identified as endosomal and more recently as a component of the ESCRT-1 complex [23
]. YGR206wp and Ylr426wp physically interact with the V-ATPase subunit Vma6p [21
]. Their corresponding genes together with YNL080C
were the only uncharacterized ORFs from our dataset that overlapped previously unknown genes whose deletion resulted in CPY secretion [15
]. Excluding the 10 dubious ORFs described above, the remaining 15 unknown genes have not been associated previously with protein sorting pathways. Among these unknowns, Yil041wp (Gvp36p) was localized to Golgi vesicles [26
]. The mutants for the unknown ORFs YDR357C
were hypersensitive to Sortin2 and their gene products were identified as interacting partners in a two-hybrid assay [22
]. The GFP fusion of Ygl079wp co-localizes with an endosome marker [23
]. In addition, Ydr357cp and Yll049wp, both from the Sortin2 dataset, physically interact with the autophagy protein Atg17p [21
] suggesting a role in the endomembrane system. Similarly, YIL039W
and YLR361C (DCR2)
genetically interact with the Golgi residents Ric1p and Ypt6p [27
], both of which mediate vesicular trafficking at the Golgi apparatus. Tvp38p (Figure ) is an integral membrane protein localized to late Golgi vesicles along with the v-SNARE Tlg2p [26
To test the interaction of mutants in unknown ORFs with Sortin2, active and inactive analogues were tested for their ability of trigger CPY secretion in the corresponding deletion mutants for YDR525W-A/SNA2, YDR105C/TMS1, YGL079W, YLL049W, YKR088C/TVP38 (data no shown). All of the mutants were more hypersensitive compared to their parental strain when grown in the presence of bioactive compounds 2 and 5. In contrast, none of the strains tested were hypersensitive to the inactive Sortin2 analogues 1, 4 and 7. Therefore, the Sortin2 hypersensitivity of mutants in these genes of unknown function was specific for analogues possessing features important for Sortin2 bioactivity. There were no detectable ALP or PGK proteins in the culture media for any Sortin2 analogs (data no shown) indicating that the CPY secretion was not due to the cell lysis.
In summary, we assigned a putative role in protein trafficking to 15 ORFs of unknown function in S. cerevisiae based on their Sortin2-induced vps phenotypes. The actual role of these ORFs will be addressed in future studies.