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

Abstract

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 (PEPAnanodiscs), 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.

Recent advances in membrane mimetics for membrane protein research

“…The water-soluble environment provided by the nanodisc is invaluable for characterizing these binding events in the absence of detergents [ 33 , 44 , 51–54 ]. ‘Empty’ lipid-only nanodiscs have also been used to monitor binding of peripheral membrane proteins onto membrane lipids [ 40 , 55 ]. This application makes nanodiscs particularly useful in the context of peripheral membrane proteins involved in signaling pathways, as their activity is often influenced by specific phospholipids [ 56 ].…”

Volleying plasma membrane proteins from birth to death: Role of J-domain proteins

“…Interestingly, Caj1 also associates with different lipids, reportedly phosphatidic acid and phosphatidylinositol-5-phosphate. Besides being majorly cytosolic, Caj1 is partially localized to the PM as well ( Dobriyal et al, 2020 ; Herianto et al, 2021 ; Zhang et al, 2021 ). Overexpression of Caj1 (but not Caj1 H34Q mutant) stabilized tryptophan permeases Tat1 and Tat2, suggesting a possible role of Hsp70:JDP machinery in regulating the stability/turnover of PM proteins in budding yeast.…”

“…Nevertheless, different Hsp70s and JDPs have been implicated in assisting PM proteins in their biogenesis, maturation, trafficking, and degradation in different organisms. Moreover, the ability of selected Hsp70s and JDPs to directly associate with lipids and membranes sheds light on their possible role in the PM as well ( Caplan et al, 1992 ; Kanazawa et al, 1997 ; Beilharz et al, 2003 ; Kalli et al, 2013 ; Radons, 2016 ; Sopha et al, 2017 ; Sjögren et al, 2018 ; Dobriyal et al, 2020 ; De Maio and Hightower, 2021 ; Zhang et al, 2021 ) ( Table 1 ). The importance of different chaperones and other PQC factors in diseases associated with PM proteins is discussed elsewhere ( Juarez-Navarro et al, 2020 ).…”