Understanding the principles of cisternal rim localization in Golgi proteins

Abstract

Type II transmembrane proteins possess a single internal hydrophobic signal–anchor sequence that functions both as an ER signal sequence and a membrane–anchor sequence. This internal signal directs the protein to be inserted in the membrane such that the N-terminal faces the cytosol, and the C-terminal faces the lumen. The Golgi complex is comprised of a series of flattened membrane vesicles (cisternae) which are surrounded by several spherical membranous vesicles. The cisternae are differentiated into distinct compartments designated as cis, medial, trans, and Trans Golgi Network. Unlike the dilated periphery (~100 nm width), the interior of the cisternae is narrow (~20 nm width) which is attributed to an array of membrane-linking proteins in a zipper-like fashion. A clear spatial separation is present between the Golgi machinery components and enzymes - Golgi enzymes are localized to the interior of the Golgi stacks, termed “the Golgi enzyme matrix”, whereas the components of the trafficking machinery are rim localized. GPP130 is a Type II transmembrane Golgi protein that localizes to the cisternal rim. It contains a cytoplasmic domain, a transmembrane domain (TMD), and a luminal domain comprising a Stem region (initial 210 amino acids juxta position to TMD) and an unstructured sequence of 451 amino acids enriched with acidic residues. Previous studies in the Lu Lab (unpublished data) have demonstrated that the stem domain of GPP130 (Gstem) is sufficient for its rim localization. However, the mechanisms guiding the localization are not known. The study aims to investigate the mechanisms for GPP130’s rim localization. The Gstem was split into two halves to test if splitting the coiled-coil structure could still lead to cisternal rim localization and it was observed that both halves were localized to the interior of the cisternae. We were interested to see if increasing the distance between the TMD and the Stem region of GPP130 can affect the localization of the construct. It was observed that with an increase in the number of amino acid residues between TMD and Gstem, the localization gradually shifts from the rim to the interior of cisternae. Studies by Mukhopadhyay et al. have suggested that Gstem is involved in the oligomerization of GPP130 in the presence of Manganese. Also, Analytical Ultracentrifugation data from the Lu Lab shows that a small population of Gstem exists as a dimer in a solution. This prompted us to test for self-interaction in Gstem. Using a pull-down assay, it was confirmed that Gstem is capable of self-interaction. In summary, the study investigates the mechanisms underlying the rim localization of GPP130 and the findings reveal that Gstem is crucial for rim localization, with observed shifts to the interior of cisternae as the distance between TMD and Gstem increases. Furthermore, the study also demonstrates the self-interacting nature of Gstem, supporting previous indications of its role in GPP130 oligomerization. These insights hold the promise to contribute to a nuanced understanding of the intricate processes governing Golgi protein localization.