Biology subjects

Prem, R.

Publications and source records attributed to Prem, R..

4 recordsLinked to original sources

bioRxiv2026

A thioester-thiol exchange reaction in Salmonella Invasion acyl carrier protein transfers the 4PP-bound acyl chain to a distal cysteine

Salmonella Typhimurium encodes two functionally distinct acyl carrier proteins: AcpP, involved in fatty acid synthesis, and the invasion acyl carrier protein (IacP), which mediates the acylation of SipB (Salmonella invasion protein B), a modification essential for SipB maturation and efficient host invasion. Despite growing insights, the IacP-dependent machinery governing acyl chain transfer remains largely enigmatic. Therefore, a multidisciplinary approach combining biochemistry and NMR was employed. A D35NIacP mutant (IacPN) that expresses in the apo-form was enzymatically converted into acyl-IacPN intermediates. Unexpectedly, the 1H15N HSQC spectra of the acyl-IacPN intermediates displayed two sets of resonances for several of the amides. The first was a less intense set of peaks, positioned close to the holo-peak, displaying a progressive chemical shift change with increasing acyl-chain length, reminiscent of the 4-phosphopantetheine (4-PP)-bound conformer of type II ACPs. The second, a more intense, acyl-chain-length-independent, previously uncharacterized set of peaks, was clustered near the longer 4PP-bound acyl-chain conformers (C14-). The origin of this new conformer was investigated by generating three cysteine-to-alanine mutants, C16A, C54A, and C69AIacPN. The C54AIacPN mutant displayed a single set of peaks in the acyl-IacPN intermediates, corresponding to the 4PP-bound IacPN conformer, establishing the critical role of Cys 54 in generating the uncharacterized peak and identifying it as a Cys-bound conformer. The acyl-S-carboxyamidomethyl-IacPN intermediates also displayed a single conformer, supporting the role of a cysteine in giving rise to the new peak. Collectively, our studies uncover a unique thioester-thiol exchange reaction in which the 4PP-tethered acyl chain is chemically transferred to a distal cysteine (Cys 54). Given that Cys 54 lies within the SipB interaction interface of IacP, this acyl chain relay process may contribute to IacP-mediated SipB acylation and Salmonella virulence.

biochemistry↗

bioRxiv2026

Djp1 is a multifunctional Hsp40 cochaperone for mitochondrial phospholipid metabolism

Mitochondria are cellular energy hubs best known for ATP production via oxidative phosphorylation; however, they also serve as biosynthetic centers for phospholipids. Mitochondrial phospholipids are critical for various cellular processes, and their loss underlies myriad mitochondrial diseases. The critical enzymes underlying these biosynthetic cascades are encoded in the nucleus, translated in the cytosol, and imported into mitochondria. Understanding of mechanisms and factors that ensure precise targeting of proteins to mitochondria has been long overlooked but remains critical. Recently, the J-protein/Hsp40 cochaperone Djp1 has emerged as a key player in mitochondrial protein targeting by promoting the transfer of precursors from the endoplasmic reticulum (ER) surface to mitochondria in a pathway termed ER-SURF. Molecular details regarding how Djp1 recognizes clients and more broadly supports mitochondrial function remain unknown. Using biochemical approaches, proteomics, and thin layer chromatography, we demonstrate that Djp1 is a regulator of Phosphatidylserine decarboxylase 1 (Psd1), an inner mitochondrial membrane resident responsible for mitochondrial phosphatidylethanolamine (PE) production. This regulation of Psd1 biogenesis is dependent on its mitochondrial targeting signal and is specific to Djp1 compared to other members of the Hsp40 family or ER targeting factors. Intriguingly, the combined loss of Djp1 and Psd1 results in a synthetic sick phenotype that unexpectedly reflects a role(s) for Djp1 in proper mitochondrial phospholipid metabolism independent of Psd1. Taken together, these findings expand our understanding of Djp1-dependent mitochondrial protein regulation and unveil Djp1 as important for mitochondrial phospholipid metabolism by multiple mechanisms.

cell biology↗

bioRxiv2026

Rewiring Mitochondrial Phosphatidylethanolamine Metabolism Identifies New and Unaccounted Trafficking Steps

The distinct compositions of the two mitochondrial membranes are generated through a combination of phospholipids that mitochondria can make and those they take; both processes depend on a series of distinct lipid trafficking steps. Mitochondria make phosphatidylethanolamine (PE) through the action of the phosphatidylserine decarboxylase Psd1, an intermembrane space (IMS)-facing integral inner membrane (IM) protein. Psd1 has been proposed to act on its endoplasmic reticulum-derived substrate, phosphatidylserine (PS), after its transport to the mitochondrial outer membrane (OM) and either following its Ups2/Mdm35-mediated transport across the IMS to the IM or instead, on the IMS-side of the OM in a process enabled by the mitochondrial contact site and cristae organizing system (MICOS). Here, we implement a two-pronged Psd1 rewiring-based strategy predicted to either 1) circumvent the need for Ups2/Mdm35 and/or MICOS; or 2) selectively ablate the ability of Psd1 to work in trans. Our results with yeast harboring Psd1 targeted to the OM demonstrate that, with respect to mitochondrial PE production, Ups2/Mdm35 and MICOS indeed function within the IMS. Using yeast expressing a topologically inverted Psd1 chimera that faces the matrix, we identify previously unappreciated transbilayer lipid trafficking steps within the IM and show that Psd1 does not operate via a MICOS-organized in trans mechanism. Further, retained flux through inverted Psd1 when both Ups2/Mdm35 and MICOS are absent strongly implicates the existence of a major, yet presently unknown, mediator(s) of lipid movement across the IMS. Collectively, these data suggest a new model of how mitochondrial membrane diversity is established and maintained.

cell biology↗

bioRxiv2025

A single mutation in the DSL motif of the acyl carrier protein can prevent its in vivo modification by E. coli Holo-acyl carrier protein synthase (AcpS)

E. coli expression system is the method of choice to obtain high yields of a pure protein. However, there is always a chance that an overexpressed protein shares structural or sequence homology with the substrate of an E. coli enzyme. In such cases, the expressed protein may be partially or fully converted into the product. A notable example is the expression of acyl carrier proteins (ACP) in E. coli. Since most type II ACPs of the fatty acid synthesis pathway (FAS) have a conserved helix II, the carrier proteins are recognized as a substrate by Holo-acyl carrier protein synthase (AcpS). Thus, most ACPs express as partially or fully loaded proteins in E. coli. This undesirable modification is a concern when the objective is to obtain milligram amounts of apo-ACP. Here, using an approach combining mutagenesis, enzyme activity, and NMR, we probed for residues in ACP that can prevent this in vivo modification, without affecting Sfp (Surfactin synthetase activating enzyme) function. Taking cues from the E. coli ACP-AcpS structure (PDB 5VCB), charge neutralization mutations were designed at five different positions in EcACP that participate in ion-pair interaction with AcpS. Three of the mutants expressed solely as apo-ACP in E. coli viz. D35N, E41A and E47A/E48A. However, only the D35N mutant could be converted into holo-/acyl-ACP using Sfp in vitro, establishing mutagenesis as a viable strategy to prevent undesired modifications in vivo. As proof of principle, the mutation was applied to two unrelated ACPs that express primarily as modified proteins in E. coli -Mus musculus mitochondrial FAS ACP (MmACP) and Salmonella Typhimurium invasion acyl carrier protein (IacP). Single D35N mutation of the ACPs prevented their in vivo modification by AcpS, and the mutants were efficiently converted into holo-ACP by Sfp in vitro. These results demonstrate that D35N mutagenesis is a useful strategy to express apo-ACP in E. coli and is applicable across all type II ACPs. Furthermore, we show that holo-IacP and holo-MmACP are not recognized as substrates by AcpH (E. coli Acyl carrier protein hydrolase), and therefore they express predominantly as modified proteins in E. coli.

biochemistry↗