The hallmark of the eukaryotic cell is its subcellular compartmentalization into specialized organelles. The structural and functional distinction of each organelle is maintained by its unique set of resident proteins. Accordingly, several mechanisms have evolved to deliver specific proteins to specific organelles. In our laboratory, studies are directed at the late steps of protein trafficking to the lysosomal vacuole of the yeast Saccharomyces cerevisiae. Yeast is the most widely used model system for study of molecular cell biology; it has served as a seminal tool in elucidating various components and pathways of trafficking in cells. The vacuole of yeast is functionally analogous to the mammalian lysosome; both play a central role in cell physiology including protein and toxin degradation, autophagy, receptor down regulation, stress survival, as well as pH and osmoregulation. Molecules destined for degradation, as well as enzymes responsible for degradation, are delivered to the vacuole by a complex series of events and molecular machinery which are subject of current and intensive study. The importance of correct lysosomal sorting is exemplified by the serious lysosomal storage diseases in humans that result from mislocalization of lysosomal proteins (e.g. I-cell disease, pseudo-Hurler polydystrophy). Additionally, cancer cells of different tissue types have been shown to specifically mislocalize a number of lysosomal enzymes. Most recently, lysosomal trafficking and function defects have been associated with various neurodegenerative diseases including Alzheimers. Several mammalian counterparts of yeast vacuolar trafficking genes have been identified, and several such counterparts have been shown to be involved in onset of diseases mentioned above. Thus, identification of new genes in lysosomal delivery, function, and biogenesis may also uncover additional genes involved in tumorigenesis and neurodegeneration.
The least defined aspect of vacuolar delivery remains its last stage where both degradative enzymes and molecules destined for degradation are transported to the vacuole from an intermediate compartment called the endosome. This step is characterized by dynamic fusion/fission events at the vacuole. Furthermore, the vacuole itself is a dynamic organelle that undergoes fusion/fission in response to various stresses and during cell cycle progression. Thus, genes regulating vacuolar fusion/fission are of interest to understanding regulation of this critical set of dynamics. The limited knowledge is due to a shortage of identified genes which regulate this step. Using a novel genomic screen approach, our laboratory identified genes involved at endosome and vacuole interface – hence named ENV genes by us. Our current focus is on ENV7 – its function, trafficking and regulation.
Through phylogenetic and bioinformatics analyses, we have also established ENV7 gene as a homolog of the human STK16 kinase whose function remains under investigation (Manandhar et al., 2013).
We have also established that the protein product of the gene, Env7 protein, is a phosphorylated and palmitoylated protein kinase localized to the membrane of the lysosomal vacuole and is involved in regulation of membrane fusion dynamics. Furthermore, we have established that in vivo phosphorylation of Env7 is dependent on YCK3, a gene encoding a yeast casein kinase (Manandhar et al., 2013,2014; Manandhar and Gharakhanian 2013). We are currently pursuing hypothesis-driven experiments to explore ENV7 as a conserved node in a proposed novel kinase cascade involved in regulation of membrane dynamics.
Our current research is supported by a major National Institutes of Health (NIH) grant to E. Gharakhanian (NIH-SCORE 1).