Marius K. Lemberg

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

We recently demonstrated that the endoplasmic reticulum export of proTGFα (encoded by TGFA) is tightly gated and that proteostasis factors, including the intramembrane protease RHBDL4 (also known as RHBDD1), can promote its release by an unknown mechanism. While investigating a potential link between RHBDL4-regulated p24 (TMED) family proteins and proTGFα trafficking, we uncovered an RHBDL4-independent pathway in which the cargo receptor TMED9 induces unconventional protein secretion (UcPS) of ectopically expressed proTGFα. This pathway selectively requires GRASP65 (also known as GORASP1), but not the closely related GRASP55 (or GORASP2), and depends on components of the autophagic machinery and the ESCRT-associated protein ALIX (also known as PDCD6IP), consistent with a Golgi-bypass secretion route. TMED9- and GRASP65-induced proTGFα secretion is insensitive to brefeldin A treatment and involves trafficking through pre-Golgi compartments. Although largely based on overexpression systems, our findings identify a previously unrecognized UcPS mechanism and provide insight into alternative protein trafficking pathways that might be exploited under conditions of proteostasis stress and in pathological contexts such as cancer and inflammation. At present, our data establish the existence of a TMED9- and GRASP65-dependent Golgi-bypass trafficking route under conditions of ectopic cargo expression, whereas its endogenous physiological relevance remains to be established.

We recently demonstrated that the endoplasmic reticulum export of proTGFα is tightly gated, and that proteostasis factors, including the intramembrane protease RHBDL4, can promote its release by an unknown mechanism. While investigating a potential link between RHBDL4-regulated p24/TMED family proteins and proTGFα trafficking, we uncovered an RHBDL4-independent pathway in which the cargo receptor TMED9 induces unconventional protein secretion (UcPS) of ectopically expressed proTGFα. This pathway selectively requires GRASP65, but not the closely related GRASP55, and depends on components of the autophagic machinery and the ESCRT-associated protein ALIX, consistent with a Golgi-bypass secretion route. Although largely based on overexpression systems, our findings identify a previously unrecognized UcPS mechanism and provide insight into alternative protein trafficking pathways that may be exploited under conditions of proteostasis stress and in pathological contexts such as cancer and inflammation.

Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimer’s disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4’s kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Förster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4’s enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4’s active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4’s catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

The toll-like receptor 4 (TLR4) is a central regulator of innate immunity that primarily recognizes bacterial lipopolysaccharide cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4 triggers degradation of TLR4’s trafficking factor TMED7. This counteracts TLR4 transport to the cell surface. Notably, TLR4 activation mediates transcriptional upregulation of RHBDL4 thereby inducing a negative feedback loop to reduce TLR4 trafficking to the plasma membrane. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling in an in vitro Mycobacterium tuberculosis macrophage infection model and consequently alleviates septic shock in a mouse model. A hypomorphic RHBDL4 mutation linked to Kawasaki syndrome, an ill-defined inflammatory disorder in children, further supports the pathophysiological relevance of our findings. In this work, we identify an RHBDL4-mediated axis that acts as a rheostat to prevent over-activation of the TLR4 pathway.

Cells need to detect and degrade faulty membrane proteins to maintain homeostasis. In this study, we identify a previously unknown function of the human signal peptidase complex (SPC)—the enzyme that removes endoplasmic reticulum (ER) signal peptides—as a membrane protein quality control factor. We show that the SPC cleaves membrane proteins that fail to correctly fold or assemble into their native complexes at otherwise hidden cleavage sites, which our study reveals to be abundant in the human membrane proteome. This posttranslocational cleavage synergizes with ER-associated degradation to sustain membrane protein homeostasis and contributes to cellular fitness. Cryptic SPC cleavage sites thus serve as predetermined breaking points that, when exposed, help to target misfolded or surplus proteins for degradation, thereby maintaining a healthy membrane proteome.

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.