Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion

ABSTRACT In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Michael J. Cohen, Brianne Philippe, Peter N. Lipke
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	cell wall disulfide reduction membrane integrity enzyme assay spheroplast Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/7398c0bbe6734ee3ac3301bb9cbe4827
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:7398c0bbe6734ee3ac3301bb9cbe4827
record_format	dspace
spelling	oai:doaj.org-article:7398c0bbe6734ee3ac3301bb9cbe48272021-11-15T15:31:13ZEnzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion10.1128/mSphere.01027-202379-5042https://doaj.org/article/7398c0bbe6734ee3ac3301bb9cbe48272020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.01027-20https://doaj.org/toc/2379-5042ABSTRACT In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells. IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.Michael J. CohenBrianne PhilippePeter N. LipkeAmerican Society for Microbiologyarticlecell walldisulfide reductionmembrane integrityenzyme assayspheroplastMicrobiologyQR1-502ENmSphere, Vol 5, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	cell wall disulfide reduction membrane integrity enzyme assay spheroplast Microbiology QR1-502
spellingShingle	cell wall disulfide reduction membrane integrity enzyme assay spheroplast Microbiology QR1-502 Michael J. Cohen Brianne Philippe Peter N. Lipke Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion
description	ABSTRACT In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 μM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells. IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.
format	article
author	Michael J. Cohen Brianne Philippe Peter N. Lipke
author_facet	Michael J. Cohen Brianne Philippe Peter N. Lipke
author_sort	Michael J. Cohen
title	Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion
title_short	Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion
title_full	Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion
title_fullStr	Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion
title_full_unstemmed	Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion
title_sort	enzymatic analysis of yeast cell wall-resident gapdh and its secretion
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/7398c0bbe6734ee3ac3301bb9cbe4827
work_keys_str_mv	AT michaeljcohen enzymaticanalysisofyeastcellwallresidentgapdhanditssecretion AT briannephilippe enzymaticanalysisofyeastcellwallresidentgapdhanditssecretion AT peternlipke enzymaticanalysisofyeastcellwallresidentgapdhanditssecretion
_version_	1718427831037329408

Enzymatic Analysis of Yeast Cell Wall-Resident GAPDH and Its Secretion

Ejemplares similares