Nanodisc-Based Proteomics Identify Caj1 as an Hsp40 with Affinity for Phosphatidic Acid Lipids

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking, and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs-small, water-soluble patches of a lipid bilayer encircled with amphipathic scaffold proteins-along with quantitative proteomics to identify lipid-binding proteins in Saccharomyces cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that the Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of combining proteomics with lipid nanodiscs to identify and characterize protein-lipid interactions that may not be evident using other methods. Data are available via ProteomeXchange with the identifier PXD027992.