Date of Degree

2-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Peter Lipke

Committee Members

Nicolas Biais

Mariana Torrente

Jason Rauceo

William Chirico

Anant Menon

Subject Categories

Biochemistry | Biology | Cell Biology | Microbiology

Keywords

Unconventional Protein Secretion, Enzymology, Yeast cell wall

Abstract

Proteins secreted to the extracellular environment play a fundamental role as signals, in metabolism, and a variety of other processes. The process of secretion through the endoplasmic reticulum and Golgi to the plasma membrane is well documented, and all cargo in this pathway contains a signal peptide. However, a variety of proteins secreted from eukaryotes lack a signal peptide and are called unconventionally secreted proteins. Here we discuss known mechanisms of unconventional protein secretion, as well as model proteins which follow characterized pathways. Additionally, we summarize the roles various unconventionally secreted proteins play outside of cells and suggest criteria for identifying unconventional secretion pathways used by proteins of interest.

The model organism S. cerevisiae is surrounded by a cell wall which contains a variety of unconventionally secreted proteins. One such protein, GAPDH, is enzymatically active in the wall. However, information about its secretion is lacking. We further demonstrate that GAPDH secretion can be studied using enzymology, demonstrate that the activity observed is not due to cell leakage. Additionally, we present a novel method to study secretion rates by inactivating external GAPDH and monitoring nascent enzyme activity on the surface. Finally, we demonstrate how to extract enzymatically active forms of GAPDH from S. cerevisiae without rupturing the plasma membrane.

We use these tools above to perform a genetic analysis of GAPDH secretion in S. cerevisiae deletion mutants. Here we report that components of the endolysosomal system (including the Rab5-GTPase Vp21, components of the CORVET and HOPS endosomal tethers, as well as proteins involved in multivesicular body biogenesis) are involved in GAPDH secretion. S. cerevisiae express 3 isoforms of GAPDH (encoded by TDH1, TDH2, and TDH3), and we report that Tdh3 colocalizes with the endolysosomal system, as well as the MVB (multivesicular body) marker Snf7. Therefore, we conclude that Tdh3 is taken up within endosomal membranes prior to export.

Relative GAPDH activity results chapter 3.xlsx (32 kB)
Relative GAPDH activity results - data from Chapter 3

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