Biochemical and Biophysical Research Communications
Phosphatidylserine-induced dissociation of the heterodimeric PstB2p/Pbi1p complex in yeast phosphatidylserine trafficking system
Introduction
Phospholipids are the major macromolecules that make up the biological membranes and play a crucial role in many aspects of cell biology. They are involved in preservation [[1], [2], [3]], pathogenicity [[4], [5], [6], [7], [8]], drug resistance [1,9,10], and signal transduction [11] depending on their physicochemical properties. In addition, the phospholipids present in the biological membranes have distinctive physicochemical properties, which is one of the important factors in determining the morphology and fluidity of membranes [12,13]. When the lipid composition changes, the organelle's shape changes and the function of its associated proteins is also impaired [14,15].
The biogenesis of lipids involves two coordinated processes, lipid biosynthesis, and lipid trafficking from the site of synthesis to the site of organelle membrane assembly [3]. Especially, phosphatidylethanolamine (PtdEtn) is a major phospholipid constituent of the mammalian cell membrane and is synthesized from phosphatidylserine (PtdSer) by phosphatidylserine decarboxylases [16]. PtdSer is produced in the ER and is transported to other organelles including endosome and Golgi by the lipid trafficking pathways [17]. In pathogenic yeast, decreased production of PtdEtn by loss of Psd is associated with an increase in the perception by the host immune system, thereby decreasing infectivity [18]. Yeast infectivity is a phenomenon that is independent of the production of PtdEtn by the Kennedy pathway, thus, proteins associated with transport of PtdSer and synthesis of PtdEtn by Psd have been recognized as potential drug targets [[19], [20], [21], [22]].
Genetic screens for the isolation of mutants defective in the trafficking of PtdSer from the ER to endosome identified the Sec14p homolog PstB2p [23], phosphatidylinositol (PtdIns) 4-kinase, Stt4p [24], and Psd2p [25]. The budding yeast, Saccharomyces cerevisiae contains two Psd proteins, Psd1p and Psd2p. Psd1p is localized in the mitochondria [26] and Psd2p is responsible for PtdEtn production in the Golgi and/or in vacuolar compartments [16,27]. They receive PtdSer from the nonvesicular transport system to convert PtdSer into PtdEtn, which is required for cell growth in a normal environment [16,23,28]. PstB2p is an essential part of this system mediating the transport of PtdSer from the ER to Golgi [23], and directly interacts with Pbi1p or Psd2p, while Pbi1p interacts with PstB2p and Scs2p [3]. The crystal structure of yeast Sfh3p, which displays high sequence identity with Sec14p, was solved in complex with PtdIns, revealing its ligand binding properties [29]. Despite these studies, it is not yet clear whether Pbi1p has an acetyltransferase activity. Furthermore, its distinctive role in lipid transport [3,28], and the stoichiometric model for PtdSer transport by the interplay of PstB2p and Pbi1p is still unclear. Thus, a detailed analysis using recombinant PstB2p and Pbi1p proteins and the PstB2p-Pbi1p complex is necessary for such an understanding.
In this study, we purified PstB2p, Pbi1p, and the PstB2p-Pbi1p complex and investigated the stoichiometric model of the PstB2p-Pbi1p complex in PtdSer transport from the ER to Golgi by using size exclusion chromatography with multi-angle static light scattering, mass spectrometry, reductive methylation, and homology modeling. We observed the following: first, PstB2p forms a homodimer, but exists as a 1:1 heterodimer in the presence of Pbi1p. Second, when PtdSer was added to the PstB2p-Pbi1p complex, PtdSer bound to PstB2p, triggering dissociation of the PstB2p-Pbi1p complex. These studies reveal a stoichiometric model of the PstB2p-Pbi1p complex during PtdSer transport from the ER to Golgi in yeast.
Section snippets
Molecular cloning of PstB2p and Pbi1p
Complementary DNA comprising residues 1–350 of PstB2p and residues 1–517 of Pbi1p were amplified from a S. cerevisiae cDNA library by PCR and cloned into pRSF and pGST2 [30] vectors in frame with an N-terminal six-histidine tag and N-terminal GST tag, respectively. The protease cleavage site of the tobacco etch virus (TEV) was inserted between the affinity tag and each yeast protein to remove the six-histidine and GST tags. Both the genes, PstB2p and Pbi1p were transformed into Escherichia coli
Oligomeric state of PstB2p, Pbi1p and the PstB2p-Pbi1p complex in solution
To investigate the stoichiometric model of the PstB2p-Pbi1p complex in the PtdSer transport system from the ER to Golgi, we set out to purify PstB2p, Pbi1p, and the PstB2p-Pbi1p complex. The binary complex (PstB2p-Pbi1p complex) consisting of PstB2p and Pbi1p could be reconstituted by co-expressing PstB2p and Pbi1p in E. coli. The PstB2p-Pbi1p complex was co-eluted from an affinity column and co-migrated on size-exclusion columns (Fig. 1A). To confirm the stoichiometry of PstB2p, Pbi1p, and the
Discussion
Based on our results, we propose a stoichiometric model of PtdSer-induced conformational changes during PtdSer transport in yeast (Fig. 4). PtdSer-free PstB2p dimerizes in solution and is recruited at the phosphatidic acid-rich region of the ER in order to accept PtdSer [3]. It is currently unknown whether Pbi1p has enzymatic activity, however it does physically interact with other lipid trafficking proteins such as Scs2p and PstB2p [28]. Because Scs2p is an integral membrane protein of the ER [
Acknowledgement
This study was supported by a grant from the National Research Foundation (NRF) of Korea funded by the Korean government (2015R1A5A1008958 and 2018R1A2B2008142) and a grant from the Korea CCS R&D Center (KCRC; 2014M1A8A1049296) to H.H.L.
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Cited by (1)
Sec14 family of lipid transfer proteins in yeasts
2021, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsCitation Excerpt :Adding PS to these complexes triggered their dissociation to monomeric forms. However, the presented functional model by Cho et al. [120] (Fig. 5C) does not correspond, in our opinion, to the available in vivo data. One of the problems encountered in deciphering the function of Pdr17p is lack of high- resolution subcellular localization data for Pdr17p and Psd2p.