Elucidating the composition and protein interactome of the Candida albicans eIF3 complex Restricted; Files Only

Abstract

Candida albicans is a fungal pathogen responsible for a large proportion of nosocomial infections, especially in immunocompromised patients. Existing as a dimorphic organism, C. albicans is capable of undergoing a morphological transition that is critical for its virulence from budding yeast cells to elongated filaments called hyphae. While the transcriptional regulation that is associated with hyphae formation is well-documented, comparatively little is known about the translational regulation underlying this process. Transcript levels do not always predict protein expression during morphological transition, which indicates that there is translational regulation occurring that is not known yet, representing a crucial gap in knowledge regarding hyphae induction. An important protein complex associated with the most regulated step of translation, initiation, is eukaryotic initiation factor 3 (eIF3), which has been observed to coordinate other translation factors and selectively bind to certain mRNA transcripts among other duties. While there are similarities between the eIF3 of C. albicans and that of other eukaryotes, the compositions vary. Haploinsufficiency of eIF3 has been documented in C. albicans, which indicates the importance of this complex for the yeast-hyphae transition. To help uncover the contribution of eIF3 to translational regulation during hyphae induction in C. albicans, this work investigated the subunit composition of eIF3 in C. albicans as well as its protein interactome in different morphological stages. Using epitope tagging of the eIF3b subunit and subsequent immunoprecipitation, eIF3 in C. albicans was determined to comprise 9 of the 13 subunits found in human eIF3. Co-immunoprecipitation and mass spectrometry analysis determined the changes in the eIF3 proteome over time and the strength of those interactions. Alterations in the composition of eIF3 during hyphae formation were discovered, suggesting the existence of sub-populations of eIF3 in C. albicans. These findings provide insight into the unknown mechanisms by which eIF3 influences translation regulation in C. albicans, an area of study that may prove invaluable in the fight against this lethal pathogen.

Table of Contents

Introduction

Methods

E. coli Miniprep

PCR Amplification and Gel Electrophoresis

PCR Purification and DNA Precipitation

Yeast Transformation

Western blot

Cell culture harvesting

Affinity Purification

Results

eIF3 was HA-tagged and isolated

Protein interactome of eIF3 changes throughout hyphae formation

eIF3 composition changes throughout hyphae formation

Discussion

References

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Palabra Clave	eIF3 Candida Hyphae
Committee Chair / Thesis Advisor	Sohail Khoshnevis, Emory University
Committee Members	Matthew Weinschenk, Emory University Homa Ghalei, Emory University

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