High confidence glycosomal membrane protein inventory unveils trypanosomal Peroxin PEX15

Abstract

AbstractTrypanosomatids are a subgroup of kinetoplastids that share the features of harboring a kinetoplast and a flagellum. Infections by these parasites pose a major concern to human health, causing diseases like Chagas disease, Human African Trypanosomiasis (HAT), and Leishmaniasis, affecting over 12 million people worldwide. Trypanosomatid parasites possess a unique peroxisome-like organelle called Glycosomes, which are essential for parasite survival. Formation and function of glycosomes depend on the cytosolic receptors and various peroxisomal membrane proteins (PMPs). These PMPs either act as Peroxin (PEX) proteins in the glycosome biogenesis or play a crucial role in facilitating the movement of solutes/molecules across the glycosomal membrane. Given the significance of PMPs, an inventory of the glycosomal membrane proteins will provide insights into their functional importance, and also aid in the identification of unknown or parasite specific Peroxins. In this study, we describe the isolation of glycosomes, its membrane protein enrichment, and mass-spectrometry-based proteomic analysis followed by protein-correlation profiling to establish a high-confidence inventory of 57 glycosomal membrane proteins, with 44 additional putative candidates. Four novel proteins from this inventory were validated, which includes two tail-anchored (TA) proteins, a homolog of human PXMP4, and a Macrodomain containing protein. Using a structure-based approach, we identified that the novel TA protein is the long-soughtTrypanosomaPEX15. Despite its low sequence similarity, it exhibits structural and topological similarities with its yeast (Pex15) and human counterpart (PEX26). We show that PEX15 is an integral membrane protein that localizes to the glycosome and interacts with PEX6. RNAi knockdown of PEX15 in bloodstream form (BSF) trypanosomes demonstrates that it is essential for glycosome biogenesis. Considering the low degree of conservation with its human counterpart, PEX15 is a promising molecular target for drug development.