Biology subjects

Jain, B. K.

Publications and source records attributed to Jain, B. K..

2 recordsLinked to original sources

bioRxiv2020

Transport mechanism of class-3 P4 ATPase lipid flippases

The P4 ATPases are a large family of membrane-embedded enzymes that use ATP hydrolysis to transport large lipid substrates across lipid bilayers. P4 ATPases differ in their cellular membrane location and their substrates. The structures of the endosome- and Golgi-localized class-1 phosphatidylserine flippases--such as the yeast Drs2 and human ATP8A1--have recently been reported, revealing a substrate binding site on the lumenal side and several transport states. However, a substrate binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases in other classes are still unknown. Here we report a systematic structural and functional study on two plasma-membrane localized, class-3 P4 ATPases that have broader substrate specificity, the S. cerevisiae Dnf1-Lem3 and Dnf2-Lem3 complexes. We have captured substrate lipids on both the exoplasmic and cytosolic sides, and we found that these two enzymes have very similar structures, consistent with their high sequence identity and redundant function. Unexpectedly, Lem3 contributes to substrate binding near the cytosolic surface. We found that the conformational transitions through the substrate transport cycle of these two class-3 enzymes match those of the class-1 enzymes, suggesting a conserved lipid-flipping mechanism among all classes of the P4 ATPases. Our study also revealed a helix-turn-helix insertion in the cytosolic P domain that is unique to the class-3 enzymes and plays a crucial role in their function. Therefore, the P4 ATPases may have retained an overall transport mechanism while evolving distinct features for cellular membrane localization, regulatory mechanisms, and transporting different lipid substrates.

biochemistry↗

bioRxiv2020

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. The regulation of membrane asymmetry is a fundamental characteristic of membrane biology, and is crucial for signal transduction, vesicle transport, and cell division. The type-IV family of P-ATPases, or P4-ATPases, establish membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is still unclear how these enzymes sort through the spectrum of lipids within the membrane to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are two chemically divergent lipids, glucosylceramide (GlcCer) and phosphatidylcholine ((PC) or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. Surprisingly, application of lipids that are poor transport substrates differentially affect PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition; thus, providing feedback onto the function of the P4-ATPases.

cell biology↗