Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

Inhibition of ubiquitination by mutations in ubiquitination machinery also rerouted Tat2p to the plasma membrane. Lem3p-Dnf1/2p are localized to endosomal/TGN membranes in addition to the plasma membrane. Endocytosis mutants, in which Lem3p-Dnf1/2p are sequestered to the plasma membrane, also exhibited the ubiquitination-dependent missorting of Tat2p. These results suggest that Tat2p is ubiquitinated at the TGN and missorted to the vacuolar pathway in the lem3 Δ mutant. The NH 2 -terminal cytoplasmic region of Tat2p containing ubiquitination acceptor lysines interacted with liposomes containing acidic phospholipids including phosphatidylserine. This interaction was abrogated by alanine substitution mutations in the basic amino acids downstream of the ubiquitination sites. Interestingly, a mutant Tat2p containing these substitutions was missorted in a ubiquitination-dependent manner. We propose the following model based on these results: Tat2p is not ubiquitinated when the NH 2 -terminal region is bound to membrane phospholipids, but if it dissociates from the membrane due to a low level of phosphatidylserine caused by perturbation of phospholipid asymmetry in the lem3 Δ mutant, Tat2p is ubiquitinated and then transported from the TGN to the vacuole.


INTRODUCTION
Phospholipid asymmetry of bilayer membranes is generally observed in the plasma membrane of eukaryotic organisms. In this phospholipid asymmetry, phosphatidylcholine (PC) 2 is predominantly distributed in the outer leaflet facing extracellular space (exoplasmic leaflet), whereas phosphatidylethanolamine (PE) and phosphatidylserine (PS) are distributed in the inner leaflet facing cytoplasm (cytoplasmic leaflet). The type 4 subfamily of P-type ATPase (P4-ATPase) seems to play an essential role to generate, maintain, and regulate phospholipid asymmetry by working as a "flippase", which translocates aminophospholipids from the exoplasmic leaflet to the cytoplasmic one in an energy-dependent manner (1)(2)(3)(4)(5).
Flippases also regulate functions of membrane proteins by changing transbilayer phospholipid Phospholipid asymmetry is involved in localization of Tat2p Tryptophan was supplemented to SDA medium at a concentration of 200 μg/ml (SDA200W) or 30 μg/ml (SDA30W), or was not supplemented (SDA-W). Standard genetic manipulations of yeast were performed as described previously (26). Tat2p   4 Escherichia coli strains DH5α and XL1-Blue were used for construction and amplification of plasmids. The lithium acetate method was used for introduction of plasmids into yeast cells (27,28).

Phospholipid asymmetry is involved in localization of
Strains and plasmids-Yeast strains used in this study are listed in When tryptophan import activity of Tat2p was estimated (Fig. 7B), the Tat2p content in the plasma membrane was determined. Cells were grown to early log phase in SDA-UW medium at 30°C. 50 OD 600 units of the cells were subjected to the sucrose gradient fractionation as described above to obtain the plasma membrane-rich fraction, which was analyzed by immunoblotting. The flippases for activation (15). We noticed that fpk1Δ fpk2Δ trp1Δ cells did not grow on YPDA, but on YPDAW (Fig. 1A). Consistently, dnf1Δ dnf2Δ Phospholipid asymmetry is involved in localization of Tat2p   8 trp1Δ as well as lem3Δ trp1Δ cells also exhibited tryptophan requirement, albeit weakly ( Fig. 1A).
Similar tryptophan requirement was also observed in drs2Δ trp1Δ and cdc50Δ trp1Δ mutants, but the phenotype seems to be milder compared to the dnf1Δ dnf2Δ/lem3Δ trp1Δ mutants, because the drs2Δ/cdc50Δ trp1Δ mutants exhibited slower growth even in YPDAW medium. Tryptophan requirement was not seen in the dnf3Δ/crf1Δ trp1Δ mutants (our unpublished results). These results suggest that the tryptophan requirement of lem3Δ and dnf1Δ dnf2Δ mutants reflects a unique function of Lem3p-Dnf1/2p. The severest tryptophan requirement in the fpk1Δ fpk2Δ trp1Δ mutant could be due to reduced phosphorylation of Drs2p, Ypk1p, or other substrates in addition to Dnf1p/Dnf2p (15,40). The dnf1Δ dnf2Δ trp1Δ and lem3Δ trp1Δ mutants exhibited clearer tryptophan-dependent growth at 18°C (Fig. 1B).
Interestingly, the tat2Δ trp1Δ mutant did not grow even in YPDAW, suggesting that the function or localization of Tat1p may be impaired at 18°C.
The cho1Δ trp1Δ mutant also exhibited tryptophan requirement for growth as reported previously (23), but it grew slowly even in YPDAW (Fig. 1A).
These results suggest that the tryptophan requirement in the dnf1Δ dnf2Δ/lem3Δ trp1Δ mutant is caused by dysregulation of Tat2p, and this was supported by the following results. (i) The tryptophan requirement of lem3Δ trp1Δ, fpk1Δ fpk2Δ trp1Δ, and cho1Δ trp1Δ mutants was suppressed by the overexpression of TAT2 (Fig.   1C). (ii) The tat2Δ mutation is synthetically lethal with the tat1Δ mutation in the trp1Δ background due to the severe uptake defect of tryptophan (39).
The lem3Δ trp1Δ mutation was synthetically lethal with tat1Δ, but not with tat2Δ ( Fig. 1D). Our strain background contains not only trp1Δ but also leu2Δ, his3Δ, met15Δ and lys2Δ mutations, but the lem3Δ mutant did not require a higher level of these amino acids for growth (our unpublished results), suggesting that dysregulation of an amino acid transporter is specific to Tat2p. whereas TAT2-3HA was non-functional in the tat2Δ mutant ( Fig. 2A and B). TAT2-GFP also suppressed the tryptophan requirement in the fpk1Δ fpk2Δ trp1Δ and cho1Δ trp1Δ mutants (our unpublished results). We also constructed the NH 2 -terminally tagged versions of Tat2p, but again they did not complement the tat2Δ mutation ( Fig. 2C). Therefore, we decided to generate antibodies against Tat2p.

Anti-Tat2p polyclonal antibodies were
Phospholipid asymmetry is involved in localization of Tat2p 9 generated in rabbits against two Tat2p peptides (N1, residues 5-21, and N2, residues 66-82) from the NH 2 -terminal cytoplasmic region of Tat2p. As shown in Fig. 3A, Tat2p was detected as a band of about 50 kDa in wild-type cells, but not in tat2Δ cells, by immunoblot assay with affinity-purified antibodies, although some cross-reactive bands were also detected especially with the anti-N2 antibodies. In the following experiments, the anti-N1 antibodies were used if not otherwise specified.  (Fig. 3C).
The fractionation profile of Tat2p was next examined in the lem3Δ and other mutants grown at 30°C. Tat2p in the PM-rich fraction was slightly decreased in the lem3Δ mutant, whereas it was clearly decreased in the cho1Δ mutant (Fig. 3D).
These results paralleled weak and strong tryptophan requirement in the lem3Δ trp1Δ and cho1Δ trp1Δ mutants, respectively (Fig. 1A). In contrast, although the fpk1Δ fpk2Δ mutant exhibited strong tryptophan requirement (Fig. 1A), Tat2p was only slightly decreased in this mutant, suggesting that the plasma membrane Tat2p might be less functional in the fpk1Δ fpk2Δ mutant. Tat2p in the PM-rich fraction was more decreased at 18°C in the lem3Δ and fpk1Δ fpk2Δ mutants ( Fig.   3D), again consistent with the growth phenotype ( Fig. 1B). The fractionation profile of Tat2p was also examined in the lem3Δ trp1Δ mutant grown in YPDA at 30°C, and it was confirmed that Tat2p was decreased in the PM-rich fraction (Fig. 3E).
These results suggest that the tryptophan requirement in the lem3Δ mutant is due to the decreased Tat2p in the plasma membrane. Tat2p that was not delivered to the plasma membrane seemed to be degraded, because the total cellular Tat2p level was decreased in the lem3Δ mutant mutations also suppressed growth defects of the lem3Δ mutant (Fig. 4A).
We next confirmed that the suppression was caused by increased Tat2p at the plasma membrane. The PM-rich fraction was isolated by sucrose gradient centrifugation as described in Fig.   3C, and Tat2p content was examined (Fig. 4B).
The vps1Δ mutation slightly increased Tat2p at the plasma membrane in the lem3Δ mutant, and the gga1Δ gga2Δ mutations substantially increased it, but to a lesser extent than the pep12Δ and vps27Δ mutations. In the vps1Δ and gga1Δ gga2Δ mutants, Tat2p might be transported to the vacuole via the TGN-to-early endosome pathway, which would not be affected by these mutations. As shown in was decreased in these mutants as estimated by sucrose density gradient fractionation (Fig. 5B).
These results suggest that Tat2p is not properly delivered to the plasma membrane due to some indirect defect in the TGN-to-plasma membrane transport of Tat2p.
Lem3p-Dnf1p is recycled from the plasma membrane through early endosomes to the TGN and back to the plasma membrane (6,43). We previously showed that Dnf1p-GFP was exclusively localized to the plasma membrane in the vrp1Δ mutant (6). Similarly, Dnf1p-GFP and Dnf2p-GFP were localized to the plasma membrane in end3Δ cells (Fig. 5C) (62), also exhibited tryptophan requirement that was not strongly exacerbated by the additional lem3Δ mutation (Fig. 5D) (Fig. 6B). However, the increase by also suppressed the tryptophan requirement in the end3Δ trp1Δ and vrp1Δ trp1Δ mutants (Fig. 6C).
These results suggest that ubiquitination of Tat2p occurs at the TGN, not at the plasma membrane, at least in end mutants. We also examined increased ubiquitination of Tat2p  in the PM-rich fraction (Fig. 3D) The partial amino acid sequence of Tat2pNT is shown in Fig. 8B. This region is highly variable among yeast amino acid permeases except conserved sequence (residues 78-85) that precedes the first transmembrane domain. Two lysine-and arginine-containing sequences, regions 1 and 2, which are separated by a serine-rich sequence (residues 34-47) were found. In the region 1, five lysine residues (10,17,20,29, and 31) had been identified as ubiquitin acceptor lysines (22), and these lysines were replaced with alanines to construct Tat2pNT 5K>A . There are also two arginine residues (11 and 19) in this region, and they were replaced with alanines to construct Tat2pNT 2R>A . In the region 2, there are two arginine residues (55 and 60) and lysine residues (54 and 66), and these four residues were replaced Phospholipid asymmetry is involved in localization of Tat2p   14 with alanines to construct Tat2pNT 2K2R>A . These three mutant proteins fused to GST were expressed and purified, and were examined for binding to the PC liposomes containing 20% PS (Fig. 8C).
Interestingly, the 2K2R>A substitutions greatly impaired the interaction: only 14.8 ± 1.1% of GST-Tat2pNT 2K2R>A was found in the top fraction compared to 71.2 ± 4.5% of the wild type. The binding of GST-Tat2pNT 5K>A was slightly impaired to 42.4 ± 9.6%, but that of GST-Tat2pNT 2R>A was not affected. These results suggest that the four basic residues in the region 2 are mainly involved in the PS-liposome binding.
We next constructed mutant tat2 genes containing these substitutions and expressed them in the tat2Δ trp1Δ mutant. Most interestingly, the tat2 2K2R>A gene failed to complement the tat2Δ mutation (Fig. 8D). This was because the plasma membrane Tat2p 2K2R>A was decreased as estimated by sucrose gradient fractionation (Fig. 8E). We next expressed the tat2 2K2R>A gene in the bul1Δ bul2Δ tat2Δ trp1Δ mutant. As shown in Fig. 8F, bul1Δ bul2Δ mutations clearly suppressed the tryptophan requirement of the tat2 2K2R>A mutant.
Taken together, these results suggest that Tat2p 2K2R>A is ubiquitinated and missorted to the vacuole, possibly because its NH 2 -terminal region does not interact with membranes.
We similarly examined the functionality of TAT2 5K>A and TAT2 2R>A mutant genes. The TAT2 5K>A mutation restored growth in the lem3Δ tat2Δ trp1Δ mutant as well as in the tat2Δ trp1Δ mutant (Fig. 8D), consistent with the previous observation that the mutated lysine residues are ubiquitin acceptor sites: this protein would be continuously transported to the plasma membrane from the TGN and would be defective in its endocytosis (22,65). In fact, Tat2p 5K>A was exclusively found in the PM-rich fraction (Fig.   8E). As shown in Fig. 8C, GST-Tat2pNT 5K>A exhibited a ~30% reduction in liposome binding. It is an interesting possibility that these lysine residues are also involved in sensing the PS level for ubiquitination of Tat2p. The TAT2 2R>A mutant behaved like wild type in tryptophan requirement for growth and subcellular localization ( Fig. 8D and E). Consistently, this mutation did not affect the binding to PS-containing liposomes (Fig. 8C).

DISCUSSION
Here we report that phospholipid flippases Although we cannot exclude a possibility that Tat2p is endocytosed at an increased rate in the lem3Δ mutant, it is not plausible, because (i) the plasma membrane-associated Tat2p level was not very different between pep12Δ and lem3Δ pep12Δ mutants (Fig. 4B), and (ii) Lem3p-Dnf1/2p were rather required for endocytosis (14).
If phospholipid flip at the TGN is generally required for the plasma membrane transport of Tat2p, the cdc50Δ/drs2Δ trp1Δ mutant should exhibit stronger tryptophan requirement, because Cdc50p-Drs2p is mainly localized to TGN/endosomal membranes (13). However, the cdc50Δ/drs2Δ mutants exhibited weaker tryptophan requirement than the lem3Δ/dnf1/2Δ mutants, taking into account slower growth rates of the cdc50Δ/drs2Δ mutants in tryptophan-rich medium (Fig. 1A). One possibility is that  To test this hypothesis, ubiquitination of Tat2p should be reconstituted in vitro with liposomes. PA, which seems not to be flipped by Lem3p-Dnf1/2p (14), is likely to exist in TGN/endosomal membranes at a level comparable to that of PS  7-nitrobens-2-oxa-1,3-diazol-4-yl; PM, plasma membrane; IMs, internal membranes.

FIGURE LEGENDS
Phospholipid asymmetry is involved in localization of Tat2p