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1.
Fig. P1.

Fig. P1. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

P4-ATPases use two sites for phospholipid selection and transport. Through a combination of mutational analysis and genetic screening, the residues indicated were identified as contributing to phospholipid specificity (red spots). The residues cluster into two gates (blue boxes), each contributing to phospholipid selection. A, actuator domain; N, nucleotide-binding domain; P, phosphorylation domain; 1-4, TM1-TM4.

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.
2.
Fig. 3.

Fig. 3. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

LL3–4 and TM4 mutations conferring edelfosine resistance alter Dnf1 substrate specificity. (A) Primary sequence alignment of LL3–4, TM4 from Dnf1 and Drs2 with the position of TM helices. (B) Topology diagram of Dnf1 TM3–4 indicates residues implicated in substrate preference based on D. (C) NBD-PL uptake by edelfosine-resistant Dnf1 mutants with mutations in LL3–4 (Left) and TM4 (Right). (D) PC/PE and PS/PC uptake ratios implicate four residues involved in substrate specificity (Phe587, Ile590, Val621, and Glu622). For all experiments, values are the mean (±SEM). Ratios less than 0 are not shown.

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.
3.
Fig. 2.

Fig. 2. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

TM3 mutations conferring edelfosine resistance alter Dnf1 substrate specificity. (A) Primary sequence alignment of TM1–2 from Dnf1 and Drs2. TM sequences are indicated below the sequence alignment. (B) Topology diagram of Dnf1 TM3 indicates residues implicated in substrate preference based on D. (C) NBD-PL uptake by edelfosine-resistant Dnf1 mutants. Substitutions at I545 decrease PC recognition by Dnf1, and substitutions at Asn550 increase PS uptake by Dnf1. (D) PC/PE and PS/PC uptake ratios describe two residues involved in substrate specificity (Ile545 and Asn550). For all experiments, values are the mean (±SEM). Ratios less than 0 are not shown.

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.
4.
Fig. 6.

Fig. 6. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

Control of Drs2 PS recognition by both cytosolic and exofacial residues. (A) DRS2 alleles harboring cytosolic cluster (exit gate) mutations complement the cold sensitivity of a drs2Δ strain. (B) Influence of exit gate mutations on PS asymmetry measured by sensitivity to PapB. Relative to WT Drs2, Drs2[F511Y] displays a defect in PS asymmetry. Drs2 N445S maintains WT resistance to PapB, whereas Drs2 N445S, [F511Y] slightly exacerbates the Drs2[F511Y] defect. (C) DRS2 alleles harboring combination entry and exit gate mutations complement the cold sensitivity of a drs2Δ strain. (D) Relative to WT Drs2, Drs2[QQ→GA] displays a major loss of PS asymmetry, which can be restored by mutation of a cytosolic cluster residue (Drs2[QQ→GA], N445S) to permit increased PS recognition. (E) Influence of cytosolic cluster mutations on PE asymmetry measured by sensitivity to duramycin. Relative to WT Drs2 and drs2∆, each mutant maintains normal PE asymmetry. Therefore, Drs2[QQ→GA] exhibits a specific defect in PS transport. (F) Combining the QQ→GA substitution with either D473K or F511Y does not disrupt PS asymmetry further or restore PS asymmetry. However, the exit gate mutation N445S can restore PS asymmetry to Drs2[QQ→GA] and Drs2[QQ→GA], [D473K]. Compare Drs2[QQ→GA], N445S with Drs2[QQ→GA] and Drs2[QQ→GA], [D473K], N445S with Drs2[QQ→GA], [D473K]. For all experiments, values are the mean (±SEM).

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.
5.
Fig. 5.

Fig. 5. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

Specificity determinants on both sides of the membrane cooperate to transport phospholipid. (A) Combination of the [GA→QQ] substitution (exofacial) with either N550S or [Y618F] (cytosolic) results in additive increases in PS uptake activity relative to either substitution alone. Combining the two cytosolic residues (exit gate) N550S and [Y618F] results in a minor increase in PS uptake, suggesting they alter the same site. For all experiments, values are the mean (±SEM). (B) Expression levels of Flag-WT Dnf1, Dnf1[GA→QQ], Dnf1 N550S, and Dnf1[Y618F] are similar. The closed arrow indicates Flag-Dnf1 protein, and the open arrow indicates a background band in Saccharomyces cerevisiae whole-cell extracts. (C) GFP-tagged WT Dnf1, Dnf1[GA→QQ], Dnf1 N550S, and Dnf1[Y618F] display similar localization patterns, polarizing to the budding daughter cell. (Scale bar: 5 μm.)

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.
6.
Fig. 4.

Fig. 4. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

Substrate preference of TM5–6 edelfosine-resistant Dnf1 mutants. (A) Primary sequence alignment of TM5–6 from Dnf1 and Drs2 with the position of TM helices. (B) Topology diagram of Dnf1 TM1–2 indicates residues implicated in substrate preference based on D and F. (C) NBD-PL uptake by Dnf1 TM5 point mutants recovered from edelfosine resistance screen targeted to TM5–6. These mutations reduced activity but did not change specificity. (D) PE/PC and PS/PC uptake ratios provide the substrate preference based on C. (E) NBD-PL uptake by Dnf1 TM6 point mutants recovered from the edelfosine resistance screen targeted to TM5–6. (F) PE/PC and PS/PC uptake ratios depict the substrate preference based on E. A single mutation (I1235F) affected the specificity of Dnf1 by enhancing PS uptake. (G) Dnf1[GA→>QQ]; Dnf1 N550K; Dnf1 N550S; and Dnf1[GA→>QQ], N550S, [Y618F] enhance PS uptake while maintaining specificity for the phospholipid headgroup and glycerol backbone. However, the Dnf1 I1235F mutation perturbs recognition of the glycerol backbone, as indicated by increased uptake of the nonsubstrate SM. For all experiments, values are the mean (±SEM). Ratios less than 0 are not shown.

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.
7.
Fig. 1.

Fig. 1. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

TM1–2 contributes to phospholipid selection. (A) NBD-PL uptake by WT Dnf1 and Dnf1[Drs2] chimeras. TM1 and TM2 contain residues involved in phospholipid selection. (B) Primary sequence alignment of TM1–2 from Dnf1, Dnf2, Drs2, and Atp8a1. Predicted TM helices are shown above the sequence alignment. Underlined residues are pairs exchanged from Drs2 into Dnf1. The colors indicate positions where mutations caused a change in specificity (red for changes altering PS recognition and green for changes altering PC recognition) or a ≥20% reduction in activity (yellow) based on D and E. (C) Topology diagram of Dnf1 TM1–2 indicates residues implicated in substrate preference based on E. (D) NBD-PL uptake by Dnf1[Drs2] ROI chimeras identifies Drs2 residues involved in PS selection (QQ) and Dnf1 residues involved in PC selection (IF and PG). (E) PE/PC and PS/PC uptake ratios provide a measure of substrate preference of Dnf1 and chimeras, independent of the overall activity or the number of transporters at the plasma membrane. The gray zones indicate a confidence interval, and values outside this interval define a change of specificity. For all experiments, values are the mean (±SEM). Ratios less than 0 are not shown.

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.
8.
Fig. 7.

Fig. 7. From: Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases.

Residues affecting the phospholipid specificity of P4-ATPases cluster into two gates. (A) Topological diagram of Dnf1 TM1–2 and TM3–4 indicating positions of residues where mutations lead to increased PS recognition (red), reduced PC recognition (green), or reduction of overall activity (yellow). These residues cluster around a phospholipid entry gate (exofacial membrane face) and an exit gate (cytosolic membrane face). (B) Dnf1 modeled on the E1 conformational state of sarco/endoplasmic reticulum Ca2+-ATPase-1 (SERCA1) (11) indicates the relative positioning of residues involved in substrate specificity. The residues cluster into two gates, where phospholipid is selected on each side of the membrane. Light blue boxes indicate the entry and exit gates, and black lines denote the membrane boundaries. A, actuator domain; N, nucleotide-binding domain; P, phosphorylation domain. (C) Dnf1 modeled on the E2-P conformational state of SERCA1 (11) highlights the conformational changes in TM1–4 during the pumping cycle driven by transfer of phosphate from ATP to the P domain. (D and E) Proposed mechanism for phospholipid transport by P4-ATPases. Phospholipid is initially selected at the entry gate, perhaps when the pump is in the E1 conformation. On transfer of phosphate from ATP to the P domain to induce the E2 conformation, TM1–2 shift up dramatically in the membrane, feasibly providing the physical force to flip the phospholipid headgroup across the lipid bilayer and feed the substrate into the exit gate. Substrate would be released from the exit gate to the cytosolic leaflet in the E2 → E1 transition.

Ryan D. Baldridge, et al. Proc Natl Acad Sci U S A. 2013 January 29;110(5):E358-E367.

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