Biomedical Engineering Reference
In-Depth Information
Chapter 4
Evaluation of Unconventional Protein Secretion
in
Saccharomyces cerevisiae
Natsuko Miura and Mitsuyoshi Ueda
Abstract
Recent development of large-scale analyses such as the secretome analysis has enabled the discovery of a
vast number of intracellular proteins that are secreted outside the cell. Often, these proteins do not contain
any known signal sequence required for conventional protein secretion. In order to avoid misidentifi cation
of such “leaked” proteins as “secreted” proteins, reconstructing the process of protein secretion is essen-
tial. Here, we describe methods for the detection of reconstructed unconventional protein secretion and
determination of regulatory proteins of secretion in
Saccharomyces cerevisiae
. We show that conjugating
target proteins with a tag-sequence and utilizing various reagents and tools can facilitate quantitative
detection of the secretion of target proteins. We expect that these methods will reveal novel unconven-
tional secretion pathways of proteins.
Key words
Unconventional secretion,
Saccharomyces cerevisiae
, Western blotting, Recombinant
proteins, Phosphoglucose isomerase, Enolase, Glycolytic enzymes, Moonlighting proteins
1
Introduction
An increasing number of unconventionally secreted proteins have
been reported to play important roles in cell-cell communication
and infection [
1
-
4
]. Among these extracellular proteins, the
number of proteins with known functions is quite small. Recently,
unconventionally secreted proteins have been reported to be the
major secreted proteins in cancer cell lines [
5
], underscoring the
urgent need to discover the extracellular functions of such pro-
teins. A number of unconventionally secreted proteins exert
“moonlighting” functions [
6
] which are unrelated to their intra-
cellular roles, making it diffi cult to predict their extracellular
properties; nevertheless, it may be possible to study their func-
tions by inhibiting their secretion. Although a few unconven-
tional secretory pathways [
7
-
9
] have been discovered, several
other proteins, their secretory pathways, and secretion-regulatory
factors remain unknown [
10
].
