ABSTRACT Kinetoplastids have essential organelles called glycosomes that compartmentalize biochemical pathways. Our ability to target glycosomes for therapeutic development is hindered by a limited understanding of the processes that regulate these organelles. Glycosomes are heterogeneous; however, the biological significance of this organelle diversity and the molecular mechanisms that regulate it are unknown. We hypothesize that glycosome specialization is a consequence of functional specialization and organelle maturation; processes that require proteins called peroxins (Pexs). To test our hypothesis, we will pursue the following aims. Aim 1: Resolve the contribution of functional specialization to glycosome heterogeneity. Leveraging our recent development of a highly sensitive flow cytometry-based platform we will define the molecular composition of glycosomes (functional specialization) by organelle sorting, mass spectrometry, superresolution microscopy and electron microscopy. Aim 2: Identify the contribution of glycosome biogenesis to organelle heterogeneity. Based on the peroxisome paradigm, we propose that the formation of preglycosomal vesicles at the ER, maturation, and proliferation of glycosomes, which involve multiple classes of preperoxisomal intermediates, can contribute to heterogeneity. We will define the preglycosomal vesicle populations in parasites using flow cytometry. During peroxisome biogenesis in yeast, Pex13 traffics through the ER via the Sec61 translocation channel. Signal recognition particle (SRP), which targets secretory proteins to the Sec61 channel binds Pex13.1 (one of two Pex13s in T. brucei; see below). We will determine how glycosome number, morphology, size and parasite viability is affected in SRP-deficient cells. Aim 3: Define the composition of glycosome protein import complexes of T. brucei. Functional specialization requires the selective import of proteins belonging to a particular pathway or process. We will define the protein composition and post- translational modifications of glycosome import complexes and determine how phosphorylation influences import complex assembly, glycosome biogenesis, and glycosome protein import. Upon completing this first of its kind systematic analysis of glycosome specialization, we will know the extent to which glycosomes become functionally specialized and will have made inroads into resolving the de novo pathway of glycosome formation in trypanosomes. Additionally, we will have defined the molecular machinery that regulates glycosome biogenesis and functional specialization. Small molecules that inhibit protein:protein interactions that mediate glycosome protein import are lethal to parasites. We anticipate that this work will reveal additional protein interactions that can be exploited for pragmatic gain and will reveal novel mechanisms that govern organelle biogenesis and specialization in eukaryotes. The flow cytometry methods developed here can be expanded to th...