Molecular mechanisms that integrate protein quality control and transport in the early secretory pathway

Fidelity of intercellular communication relies on the specificity of receptor-ligand interactions: thus, both must have their native conformation to convey the desired signals. Most receptors and ligands are plasma membrane and secreted proteins: as such, and as about one-third of the human proteome, they have their cradle in the early secretory pathway (ESP), the functional unit formed by the endoplasmic reticulum (ER), intermediate compartment (ERGIC) and cisGolgi. Dozens of chaperones populate ESP and most bear KDEL-like motifs. These are recognized by KDEL receptors (KRs) in the Golgi and retrieved to the ER via COPI vesicles [1]. Three different KRs homologues exist in vertebrates. Moreover, KDEL-bearing proteins cycle at different rates: ERp44 does so much faster with respect to PDI, which has crucial functional consequences in IgM biogenesis [2-4]. Our preliminary data indicate that other ESP residents cycle with intermediate rates. Our proposal investigates the molecular mechanisms that promote protein structural maturation in ESP, restricting transport to only native conformers (Quality Control). We surmise that specific chaperone-KRs interaction can generate different recycling rates creating chaperones/folding enzymes gradients alongtheu ESP. Such gradients could assist the variegated human secretome and be rearranged to optimize release of tissue-specific cargoes. Moreover, the arrival of different chaperone waves binding the KRs with different rates and compositions may impact the intracellular signaling properties of KRs [5-10]. In this framework, we recently discovered the first non-peptide compounds (hereafter indicated as D11 and D17) able to bind KRs and activate or inhibit their signals (unpublished results), with wide potential applications. To obtain an unbiased view of the ensemble of ESP residents, we will analyze the dynamic changes of the secretome upon acute small molecule-mediated or genetic inhibition of the three KRs, alone or in combination. The rationale of this endeavor is threefold: i) the sooner a protein appears extracellularly, the faster is its recycling; ii) the final localization of a protein depends on the ratio between ER exit and retrieval; iii) the three KRs might differ in terms of traffic control and signaling outputs. We expect to identify groups of proteins, possibly with shared functions, that recycle at distinct rates. At least one protein in each group will be tagged with Halo to perform visual pulse chase experiments [3]. Proteomic analyses will be complemented by structural modeling of the KRs luminal interactions with either client proteins or small-molecule regulators. Deciphering the molecular machines that couple efficiency and fidelity, maintaining homeostasis in the exocytic pathway is of paramount importance, particularly in the progression of secretory tumors and in the production of recombinant proteins.