Elucidating the composition and protein interactome of the Candida albicans eIF3 complex Restricted; Files Only
Jiang, Simon (Spring 2023)
Published
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
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