טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKaiss Mohammed
SubjectStudying the role of NAC and OM14 in protein import to
mitochondria
DepartmentDepartment of Biology
Supervisor Professor Yoav Arava
Full Thesis textFull thesis text - English Version


Abstract

Mitochondria are organelles in eukaryotic cells that have many functions for which they require 1300-1500 proteins. The majority (~99%) of these proteins are encoded by nuclear DNA and only after their translation, or during translation in the cytosol, they enter into mitochondria. Previous studies provided many evidences for two possible pathways for import into mitochondria: Post-translational import?according to which, the protein is fully translated in the cytosol and is imported into the mitochondria only after its complete synthesis; and co-translational import? according to which, proteins enter the mitochondria during their translation. Recent work identified interaction between a mitochondrial outer membrane protein (OM14) and a ribosome associated complex (NAC). This interaction has been shown to support the co-translational import model. My project is to study the role of OM14 in this interaction with two specific goals: a) to determine which domains of OM14 support this interaction; and b) to identify other proteins involved in the interaction between ribosomes and mitochondria. For the first goal, I cloned OM14 and generated constructs in which I deleted each one of its cytosolic domains. Phenotypic screening for these constructs revealed differences in growth under respiration inducing conditions. However, no significant difference in the amount of NAC associated with mitochondria was detected. For the second goal, double knockout strains for OM14 and candidate receptors were generated. The double deletion strains were subjected to growth on various carbon sources. Surprisingly, I found that the double knockout om14Δpor2Δ grew better than its single knockouts. Interestingly, double knockout for om14Δpor1Δ did not show similar phenotype. This suggests a different role for Por2 and Por1. To understand the mechanism for this differential phenotype, RNA analysis was performed, and revealed upregulation of three chaperones. These may regulate protein homeostasis in om14Δpor1Δ. Interestingly, genes that are involved in metals homeostasis are at higher level in por2Δ. Mass spectrometry analysis for metal levels in W.T and por2Δ reveal differences in iron and zinc among strains.

In this study I tested the effect of OM14 cytosolic domains on its interaction with NAC, and roles of OM14 protein partners. Por2 showed genetic interaction with OM14. Moreover, preliminary results showed metals specificity for yeast mitochondrial porins. Future experiments could reveal the role of the mitochondria in Zinc homeostasis in the cell.