Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

Abstract

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyze lysosomes and early endosomes. Based on the identification of 5376 cross-links, we investigate protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and a heterodimeric structure of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identify >300 putative cargo proteins and confirm eleven substrates for flotillin-dependent endocytosis, including the latrophilin family of adhesion G protein-coupled receptors.

Proteomic Investigation of Differential Interactomes of Glypican 1 and a Putative Disease-Modifying Variant of Ataxia

“…Glypicans are mainly located at the plasma membrane, and GPC1 has been shown to be recycled via the endolysosomal pathway. After GPC1 internalization by caveolin-mediated endocytosis, it accumulates in RAB7-positive late endosomes, where its HS chains are modified and cleaved to generate anhydromannose (HS-anMan), which can interact with other molecules. , Consistent with the recycling pathway of GPC1, RAB35 and RAB10 were enriched in our dataset as recycling endosome markers. − In addition, we detected FLOT1 and FLOT2, which are located at the plasma membrane, caveolar membranes, endosomes, and lysosomes and are ascribed major roles in vesicular trafficking . Interestingly, we also identified components of the ESCRT-I (VPS28) and ESCRT-III (CHMP1A, CHMP2A, CHMP2B, CHMP4B, and CHMP5) complexes.…”

“…49,50 Consistent with the recycling pathway of GPC1, RAB35 and RAB10 were enriched in our dataset as recycling endosome markers. 51−53 In addition, we detected FLOT1 and FLOT2, which are located at the plasma membrane, caveolar membranes, endosomes, and lysosomes 54 and are ascribed major roles in vesicular trafficking. 55 Interestingly, we also identified components of the ESCRT-I (VPS28) and ESCRT-III (CHMP1A, CHMP2A, CHMP2B, CHMP4B, and CHMP5) complexes.…”

“…57,58 Regarding lysosomes, we detected several members of the vacuolar ATPase (V-ATPase) (Figure 3f), a MDa complex consisting of a membrane-embedded proton pumping V0 domain and a cytosolic ATP hydrolyzing V1 domain. 54 For both domains, we identified several subunits (V1 domain: ATPV1A, ATPVB2, ATPVC1, ATPVD, ATPVE1, ATPVF, ATPVG1, and ATPVH; V0 domain: AT6V0A1, AT6V0A2, AT6V0A3, AT6V0C, and AT6V0D1) as well as the accessory subunits ATP6AP1 and ATP6AP2, implying efficient enrichment of the intact complex. V-ATPases, whose subunits are highly enriched in our dataset, facilitate acidification/pH regulation of various intracellular compartments 59,60 and localize, e.g., to the plasma membrane, to the Golgi apparatus, and to endosomes and lysosomes.…”

Small molecules targeting endocytic uptake and recycling pathways

“…This functional module includes clathrin (both light and heavy chains), caveolin and flotillin, each of which is recruited to both plasma membrane and endosomes by several endocytic adaptor subsets. Specific protein-binding motifs enable both clathrin polymerization and caveolin/flotillin oligomerization into higherorder structures (i.e., basket, disk-shaped or tetramers, respectively), thus helping the budding of vesicles from donor membranes (Van Jaarsveld et al, 1981;Chaudhary et al, 2014;Kononenko et al, 2014;Watanabe et al, 2014;Shvets et al, 2015;Han et al, 2020;Porta et al, 2022;Singh et al, 2022).…”

Innovation in Cross-Linking Mass Spectrometry Workflows: Toward a Comprehensive, Flexible, and Customizable Data Analysis Platform

“…Chemical cross-linking combined with mass spectrometry (XL-MS) has matured into a powerful tool for validating the prediction and modeling of protein structure obtained using well-established biophysical techniques 1−3 (e.g., X-ray crystallography, nuclear magnetic resonance (NMR), cryo-electron microscopy (cryo-EM)) and computational biology techniques 4−6 (e.g., Alphafold2 and Meta AI). In addition, it complements the dynamic changes 7,8 in proteins that cannot be detected by the above techniques and captures the landscape of protein−protein interactions (PPIs). 9,10 The overall workflow of XL-MS typically consists of five steps: 11 (A) incubation of native proteins or protein mixtures with cross-linkers, (B) digestion of proteins into peptides, (C) mass spectrometry data acquisition, (D) interpretation of cross-linked data, and (E) application (study of protein structures and protein−protein interactions).…”