Ypq1 software

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Click "Literature Details" to view all literature information for this locus, including shared literature between genes. Isolation of constitutively degrading Ypq1 mutants. Vec, Empty vector. Pgk1, loading control.

FL, full-length. C Predicted structure of Ypq1 showing PQ motifs and charged residues within the translocation tunnel. In top and bottom views, loops have been removed for clarity. D General architecture of eukaryotic PQ-loop family members.

Ypq1 is a member of the PQ-loop transporter family, which has two conserved PQ motifs. Other family members include bacterial and plant sugar transporters i. Eukaryotic PQ-loop proteins typically have seven transmembrane helices Fig. Within each THB, helices are arranged in an alternating topology.

Within the PQ-loop family, several crystal structures have been solved Han et al. Using homology modeling Waterhouse et al. Through evolutionary covariation analysis Kamisetty et al. When plotted on the predicted Ypq1 structure, conserved residues in the transmembrane domains clustered together, validating the model Fig.

S1 B ; Ovchinnikov et al. Of note, the cytosolic loops, especially the large loop between TM3 and TM4, appear to be unstructured due to insufficient sequence homology within these regions Fig. Ypq1 homology model. Related to Fig. Ypq1 model cyan shown in ribbon representation. Highlighted in surface representation are negatively charged residues purple and the single positively charged residue blue facing the translocation tunnel.

Shown also are the Pro red and Gln yellow residues in the PQ motifs. Gray boxes correspond to transmembrane helices, and black connecting lines correspond to loops. B Conserved residues mapped on the homology model of Ypq1. Further examination revealed several highly conserved charged residues within the translocation tunnel i. Collectively, these make the tunnel interior negatively charged, which could be important in the transport of the positively charged lysine Yu et al. We wondered what would happen if we introduced more negative charges in the tunnel, because lysine withdrawal would also make the tunnel more negative.

To this end, we substituted noncharged residues with Asp. The mutants likely did not have gross defects in folding, or they would have been retained in the ER and degraded by ER-associated degradation pathways Ruggiano et al. Instead, they still trafficked normally to the vacuole membrane, where they were recognized by Ssh4.

In summary, we isolated three Ypq1 mutants that are constitutively degraded even in the presence of lysine. This degradation is still dependent on Ssh4.

The isolation of constitutive Ypq1 mutants enabled us to identify critical residues important for Ypq1 degradation. Because these mutants were still recognized by Ssh4, we hypothesized that they may adopt a state similar to WT Ypq1 after lysine starvation.

One testable prediction is that interrupting the Ypq1-Ssh4 interface should block both the constitutive degradation and the lysine withdrawal—triggered degradation.

To test this, we designed a suppressor-screen strategy. A suppressor screen identified clusters on Ypq1 that are important in its degradation. Vac, vacuole; Ura, uracil. B Summary of the genuine suppressor residues and their corresponding mutations.

H Critical regions on Ypq1 based on suppressor screen mapped on the conserved architecture of PQ-loop proteins. I Critical residues mapped on the 3D model of Ypq1. TM4 shown as a reference point. To ensure stringency, we overexpressed Ssh4 under the control of the CYC1 promoter.

Emerging suppressors that effectively block the constitutive degradation would stabilize the fusion protein on the vacuole membrane, thereby allowing yeast to survive on media lacking uracil.

Using this strategy, a total of 99 unique suppressor mutants were recovered and sequenced Table S1. Each mutant contained a combination of one to four interspersed mutations. To determine genuine suppressors, we focused on those that either occurred as a single point mutation or appeared frequently.

Using these criteria, we identified 25 critical residues Fig. When mapped on the protein sequence of Ypq1, these residues form five distinct groups: 1 residues that affect ER exit 26 mutants , 2 the PQ motifs 9 mutants , 3 the cytosolic loop between TMs 1 and 2 8 mutants , 4 TM5 21 mutants , and 5 TM7 21 mutants , or a combination of these regions 5 mutants.

From this screen we also found two mutants IT,GD and QR,ND whose mutations did not fit into any category, and seven weak suppressors that survived the uracil selection but still appeared to localize in the lumen Table S1. For the first set of mutants, survival was due to a trafficking defect. The remaining nine ER-trapped mutants had a significant reduction in protein levels data not shown , likely due to folding defects, and so were trapped by ER quality control systems.

Although interesting, these ER-trapped mutants do not provide further insight for our study and so were not pursued further. Meanwhile, mutations on the remaining four clusters i. To test whether these regions also govern lysine-mediated degradation, we introduced these mutations into WT Ypq1-GFP. Among those we tested, imaging analysis showed that all mutations consistently blocked the sorting of Ypq1-GFP into the lumen after lysine withdrawal Fig.

These results strongly support the idea that the constitutive mutants and lysine-starved Ypq1 are using the same regions to regulate recognition by Ssh4. Next, we sought to quantify the degradation defects of these suppressors by developing a GFP-based flow cytometry method. Although GFP is resistant to vacuolar proteases, it partially drops in fluorescence under acidic pH Patterson et al. Consistently, we observed a quantifiable shift in fluorescence after lysine withdrawal when Ypq1-GFP is sorted into the acidic vacuole lumen Fig.

S2 C , indicating that the decrease in fluorescence was Ssh4-dependent. S2, D and E ; see Materials and methods for details.

Suppressor residues and flow cytometry-based method to quantify Ypq1-GFP degradation. A Workflow of flow cytometry—based degradation assay. Shown are several degradation controls deg ctrl. D Heat map showing fluorescence arbitrary units in three replicates of no GFP control, negative control, and positive control in two experimental setups.

Darker colors correspond to higher fluorescence. E Step-by-step calculation used to generate heat map in Fig. First column: Fluorescence values from three replicates were averaged. Second column: Fluorescence values were corrected by subtracting average fluorescence values of no GFP control strain. We hypothesized that the disruption might be due to Ypq1 losing its recognizability to Ssh4, presumably due to a loss of interaction. Despite many attempts, coimmunoprecipitation coIP had been unsuccessful under native conditions, likely due to the low expression of Ssh4 Kulak et al.

No coIP was observed even after overexpressing Ssh4 Fig. To mitigate these difficulties, we overexpressed Ssh4 under the control of the CYC1 promoter.

IP, immunoprecipitation. We then tested if the suppressors could be influencing the ability of Ypq1 to associate with Ssh4. Using the same coIP conditions as in Fig.

Cytosolic interactions have also been robustly studied for other Rsp5 adaptors and their cargo Crapeau et al. However, the prevalence of TM5 or TM7 mutations in our screen i. Therefore, we hypothesized that TM5 and TM7 could comprise a binding pocket for the single transmembrane helix of Ssh4. Ypq1 homology model with suppressor residues.

Ypq1 model gray shown in ribbon representation. To confirm the importance of TM5 and TM7, we performed scanning mutagenesis by mutating each residue to Ala. Using flow cytometry, imaging, and Western blot analyses, we tested whether they could block lysine-mediated degradation. As a control, we did the same scanning mutagenesis on TM3, which did not have suppressors based on our screen.

Through this scanning, we identified additional suppressor mutations in all transmembrane helices tested. S4, A and B ; and Fig. S4, D and E. In contrast, only 2 out of 20 residues in TM3 showed a strong block Fig. When imaged, the high FR mutants consistently localized on the vacuole membrane Fig.

Mapping the Ala scanning suppressors on helical wheels showed that TM5 seemed to have a cluster of important residues at one region and several scattered residues Fig. TM7 had more critical residues, and most of them line one face of the helix. Although we do not know the role of the two suppressor residues in TM3 yet, here we underscore the importance of TM5 and TM7 in mediating Ypq1 degradation, presumably by forming binding sites with Ssh4 within the lipid bilayer.

A—C Heat map showing the degradation defect of all Ypq1 mutants. WT and LA are nonblocking controls. WT and FA are nonblocking controls. The transmembrane helix of Ssh4 is important for Ypq1 degradation.

A Conceptual model of competition assay. Full-length Ssh4 can interact with and ubiquitinate Ypq1, whereas Ssh4 NT can only interact but not ubiquitinate.

Ub, ubiquitin. Vec, empty vector. Also see Fig. S4, D and E , for blots. Degradation-blocking mutants are noted as "Hits. G Predicted structure of Ssh4 TM showing critical residues red. Next, we looked into the role of Ssh4 in Ypq1 degradation. Ssh4 is a residue, type I transmembrane protein localized on the vacuole membrane.

It has a lumenal N-terminal domain that is predicted to be 46 residues long, followed by a residue transmembrane helix residues 47—69 and a large residue cytosolic region. While it is likely that Ssh4 simultaneously uses several regions to recognize Ypq1, we wanted to test the possibility that its transmembrane helix plays a functional role. First, we designed a competition assay Fig. We constructed Ssh4 NT , a truncated version that contains only the N-terminal tail and the transmembrane helix.

We retained the N-terminal tail because it contains the signal peptide that directs the protein into the secretory pathway. Using this arrangement, Ssh4 NT localized properly on the vacuole membrane Fig. We reasoned that if the transmembrane helix of Ssh4 interacts with Ypq1, then Ssh4 NT should compete with endogenous Ssh4. These data support a model wherein Ssh4 uses its transmembrane helix to interact with Ypq1. Expression and localization of Ssh4 mutants. Quantification shown in Fig.

To further test the importance of the transmembrane helix, we performed Ala scanning mutagenesis and measured the impact on Ypq1-GFP degradation. We ruled out the possibility that F55A and V63A caused a strong block because they are not expressed or are mislocalized. By Western blot and imaging, we confirmed that these mutants expressed to near-WT levels and localized on the vacuole membrane Fig.

S5, C and D. Therefore, F55 and V63 are indeed important in recognizing Ypq1-GFP, and disrupting these residues would block its degradation. We also performed Trp scanning, a common strategy used to define helical packing interfaces in membrane proteins Hong and Miller, ; Sharp et al. Compared with Ala, a bulky hydrophobic residue such as Trp would more effectively disrupt tight transmembrane helix packing between protein complexes Lemmon et al.

These last mutations conferred a block in degradation likely because they were unstable. Thus, it is inconclusive if these particular residues are critical for Ypq1 recognition. Regardless, our results strongly support the importance of the Ssh4 TM in recognizing Ypq1. We noticed a periodicity in the positions of intolerant residues, that is, strong defects occurred three or four residues apart from each other.

When mapped on a helical wheel and a 3D model, this periodicity becomes more evident as the critical residues cleanly segregated to one face of the TM Fig. Ssh4 has been implicated in vacuole membrane quality control, recognizing mislocalized plasma membrane proteins, such as the cell wall integrity sensor Wsc1, for degradation Sardana et al. S6, A and B , whereas double mutants conferred a more robust block Fig.

This suggests that the TM helix of Ssh4 is also important for recognizing other membrane proteins. S5, G and H ; and Fig. This suggests that Ssh4 potentially uses different sets of residues on its TM helix to recognize various cargoes.

G Helical wheel showing the position of residues conferring partial degradation block when mutated to Trp. Together, our results support an important role for the Ssh4 TM in mediating possible helical packing interactions with Ypq1 and other targets. We mutated one residue at the putative interface on Ypq1 to Asp, and the opposite residue on Ssh4 to Arg Fig. Due to their charged side chains, Asp or Arg could potentially perturb the hydrophobic binding interface Brender and Zhang, However, simultaneous introduction of both Asp and Arg would be tolerated by forming a salt bridge, provided that both residues are sufficiently close to each other, i.

Charge complementation pairs support a transmembrane interaction between Ypq1 and Ssh4. A Conceptual model of charge complementation. EV, empty vector; OE, overexpression. E Cell counts from D. We tested different combinations of critical residues identified by the suppressor screen and scanning mutagenesis. Using flow cytometry Fig. Remarkably, coexpressing both mutants partially restored the degradation, bringing the FR score to This complementation was further enhanced by overexpressing Ssh4 S52R , which brought the FR score to The crux of this assay is that charge complementation would only occur if the mutated residues were close to each other.

Therefore, these results suggest that Ypq1 Y can come in contact with Ssh4 S52 only when lysine is not present, possibly due to an increase in accessibility. Furthermore, increasing Ssh4 S52R levels somehow also increases the chance to access these sites.

We also mutated I53, an adjacent residue, to Arg. Cells were fixed and examined by fluorescence microscopy. Integral membrane proteins of lysosomes in animal cells, of tonoplasts in plant cells and of the vacuole in yeast follow the secretory pathway and are sorted from the Golgi to the destination membrane after packaging into vesicles.

A crucial step in this sorting is recognition, by specialized adaptor proteins or complexes 15 , of the dileucine- or tyrosine-based motif typically harbored by the proteins to be sorted. To date, however, the trafficking of yeast vacuolar transporters has been little investigated.

Here we report that the Ypq transporters proposed to mediate export of basic amino acids stored in the vacuole 6 are also targeted to the vacuole in a manner dependent on the AP-3 adaptor complex. Our data suggest that this complex recognizes an acidic dileucine motif DxxLL present in the second cytosolic loop of each of these proteins.

We find that when this dileucine motif is mutated or the AP-3 complex is nonfunctional, the Ypq proteins tend to accumulate in the Golgi but are also rerouted to endosomes. The Ypq1 and Ypq2 proteins reaching the endosomes are efficiently delivered to the vacuolar membrane. In contrast, the Ypq3 transporter tends to be missorted to the vacuolar lumen, indicating that Ypq3 deviated to endosomes is efficiently sorted via the MVB pathway into vesicles budding into the endosomal lumen, probably because Ypq3 undergoes ubiquitylation How Ypq proteins that fail to use the ALP pathway are rerouted to endosomes remains undetermined.

This sorting likely involves their packaging into vesicles thanks to alternative adaptors, e. This suggests that these adaptors function redundantly or that others promote this sorting to endosomes.

Previous work has shown that an acidic dileucine promotes APdependent sorting of the Pho8, Vam3 and Sna4 proteins 24 , 29 to the vacuole, whereas a tyrosine-based motif plays a similar role for vacuolar targeting of Sna2, Yck3 and Atg27 23 , 25 , Interestingly, the Sna2 protein contains a second tyrosine-based motif promoting APdependent sorting to the vacuole When the interaction with AP-3 is impaired, these proteins undergo different fates: proper sorting to the vacuole via the alternative CPY pathway 20 , 21 , 26 , missorting to the vacuolar lumen via the MVB pathway 24 , or rerouting to the plasma membrane Studies on plant cells have likewise revealed an important role of the AP-3 complex in sorting membrane proteins including transporters to the tonoplast The AP-3 complex also mediates sorting of several proteins eg.

We now report that the PQLC2 transporter also needs the AP-3 adaptor complex to be properly targeted to lysosomes in HeLa cells, as well as to the vacuole when produced in yeast.

A recent study has shown that sorting to the lysosome of cystinosin, the cystine exporter of the PQ-loop family, involves a tyrosine-based motif interacting with the AP-3 complex. If this interaction is impaired, cystinosin tends to be targeted to the plasma membrane Furthermore, when produced in yeast, cystinosin is delivered to the vacuolar membrane but is partially deviated to the cell surface if the AP-3 complex is deficient our unpublished data.

These data further support the conclusion that yeast and mammalian PQ-loop transporters traffic to the vacuole or lysosome via similar APdependent mechanisms. However, the AP-3 complex of yeast is thought to recruit cargoes only at the trans-Golgi for direct targeting to the vacuole whereas that of mammalian cells operates at early endosomes where it appears to sort proteins to deliver them to late endosomes, lysosomes or lysosome-related organelles, ie.

The basic role of AP-3 in yeast and mammalian cells could therefore be to circumvent possible delivery of cargoes to the lumen of these compartments 14 , a model supported by our observation that Ypq3 and PQLC2 produced in yeast are targeted to the vacuolar lumen when APmediating sorting is defective.

Interestingly, a recent study reported that the Ypq1 transporter present at the vacuolar membrane can subsequently be targeted to the vacuolar lumen.

This occurs under lysine-starvation conditions and requires prior ubiquitin-dependent sorting of Ypq1 to endosomes where the transporter is sorted into intraluminal vesicles It will thus be interesting to determine whether other lysosomal transporters unrelated to the PQ-loop family also use a dileucine- or tyrosine-based motif recognized by the AP-3 complex to reach their destination membrane circumventing the multivesicular body pathway and if they can subsequently be targeted to the lumen of the lysosome via a pathway similar to the one described for Ypq1 in yeast.

The plasmids used in this work are listed in Table 2. The functionality of the Sec7-mCherry construct was verified by testing its capacity to complement a thermosensitive sec7 mutant see Supplementary Fig.

S8, online. Experimental details, including the sequences of the oligonucleotides used in PCR reactions for the isolation of yeast mutant strains and plasmids, are available upon request. Labeling of the vacuolar membrane with FM was performed as described previously Quantification of the subcellular location patterns of Ypq-GFP proteins was based on images obtained in three independent experiments. For each experiment, one hundred randomly-selected cells were examined by eye and classified in different subcellular localization patterns: vacuolar membrane, vacuolar membrane and several dots, cytoplasmic distribution and dots, a combination of cytoplasmic distribution, dots and vacuolar membrane, and vacuolar lumen and dots.

The cells were transfected with 0. AllStars negative control siRNA was used as a control mock. Cells were seeded at 1.

The samples were then boiled in sample buffer, run on an SDS polyacrylamide gel, and subjected to western blotting. The blot was probed with anti-AP3M1 antibody Abcam, ab How to cite this article : Llinares, E.

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