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October 16, 2003
Researchers Create New Atlas of Yeast Proteins
Using high-tech robots and old-fashioned hard labor, Howard Hughes
Medical Institute researchers have measured the abundance and
pinpointed the cellular locations of more than 4,000 proteins in
yeast.
Proteins are the workhorse molecules of the cell. They catalyze
reactions, transport molecules within the cell and switch genes on and
off. Measuring the abundance of and identifying the cellular locations
of yeast proteins will be invaluable in helping to understand the
complex biology of a relatively simple organism. Beyond that, however,
the effort is emblematic of a shift in biological research, toward
understanding how changes in the “proteome” — the
interacting global network of proteins in a cell — can influence
cellular “behavior.”
“Now, investigators can measure the abundance of proteins and follow their location with a degree of sensitivity that was never possible for any proteome in any organism.”
Jonathan S. Weissman
Research teams led by HHMI investigators Erin K.
O'Shea and Jonathan
S. Weissman, both of the University of California at San Francisco,
published two research articles describing their work in the October
16, 2003, issue of the journal Nature.
“We have now made the yeast proteome accessible in a way it
simply wasn't before,” said Weissman. “Now, investigators
can measure the abundance of proteins and follow their location with a
degree of sensitivity that was never possible for any proteome in any
organism. We believe that this capability really strengthens the status
of yeast as the premier organism for the systems-biology approach to a
coherent, comprehensive understanding of how the cell works.”
According to the researchers, determining the relative abundance of
proteins in yeast will offer more insight into protein function than do
studies of levels of messenger RNA (mRNA), the most widely used
indicator of cellular activity. Messenger RNA molecules are the genetic
templates for proteins. In constructing proteins, the mRNA template is
transcribed from genes and transported to the ribosomes - the cell's
protein “factories” that are large complexes of protein and
RNA.
“The basic problem is that, in the end, all the cell's
functions are carried out by proteins, and proteins are encoded by
messenger RNA,” said Weissman. “But changing the mRNA level
is only one of many mechanisms that the cell uses to alter levels of a
protein.” For example, the cell may produce and process proteins
in numerous ways that ultimately affect the composition of the working
molecule and its abundance. “The most abundant level of mRNA in a
yeast cell is about a hundred molecules, and the lowest, non-zero level
a cell can have is, by definition, one,” he said. “But the
functional level of protein in a cell can vary enormously, as much as a
million-fold difference.” Thus, measuring the levels of proteins
in the cell is critical to understanding the functional properties of
proteins.
Likewise, mapping the location of individual proteins in the cell is
also critical to understanding protein function, said O'Shea. “A
major goal of genomics and proteomics is to understand the function of
each protein, and important clues can be gleaned from where each
protein is within the cell,” she said.
To measure the levels and locations of the thousands of individual
yeast proteins, the researchers developed a method for tagging each
protein. Without a common tag, measuring individual proteins is all but
impossible, given that there are so many and each protein is
biochemically unique. So, the researchers employed a widely used
technique to introduce the DNA for a specific tag into each of the
genes that specify each yeast protein. To create the targeted tags,
they synthesized about 13,000 gene sequences that would target one of
the tags to the end of each gene in the yeast genome. They used these
specific tags to mark some 6,234 yeast gene segments, called
“open reading frames.”
As a result of the gene-tagging, the researchers created two
different “libraries” of yeast cells. One library consisted
of roughly 4,200 yeast ”strains,” each producing one tagged
protein — tagged with a sequence that makes it easy to detect. This
method permitted the researchers to use antibodies to quantify the
level of protein in the cell. The other library, used for protein
localization studies, consisted of strains where each protein is tagged
with a sequence that produced a green fluorescent protein visible under
a microscope.
“Fortunately, yeast cells have a peculiar property that
enables us to target gene insertions to specific points on the
chromosome,” said O'Shea. “So, part of what's special about
these experiments is that we could measure proteins that are expressed
from the chromosome under normal control. So, they are expressed at
physiologically relevant levels and in relevant places in the
cell.”
The researchers' protein-expression studies revealed the abundance
of some 4,251 proteins in the yeast cell. “The experimental
highlights for me are, first, that we can detect more than eighty
percent of the yeast proteins in the cell — and that's a high fraction
of the genome to be expressing at one time,” said O'Shea.
“And equally interesting is that we can see a vast range of
protein abundance, from fewer than fifty molecules per cell to more
than one million.” In contrast, previous methods of detecting
yeast proteins identified only the more abundant proteins, missing rare
but critical proteins, such as those that switch on genes.
“We just didn't know how much of the proteome the cell needed
during growth,” said Weissman. “We might have guessed that
the cell had many genes in reserve for other purposes. So, the eighty
percent expression level we detected was a bit of a surprise. Also,
although we had hints that proteins existed at a wide range of
abundances, until these measurements were made, there was no way to
quantify that range.”
In addition to identifying many thousands of functional genes, the
researchers also quantified the number of “spurious open reading
frames,” which are DNA segments that appear to be genes, but
which are not. Their findings, they said, agreed with previous studies
of nonfunctional DNA segments by other researchers.
The researchers next plan to use their protein libraries to explore
how protein levels change over time. Obtaining this kind of dynamic
information will be critical to efforts to model the action of proteins
as the cell grows and adapts to changing conditions. Such models will
give biologists the scientific equivalent of a movie of the cell's
machinery, rather than the snapshots available today, they said.
“Although it is interesting to know under standard lab
conditions how much of each protein is present, what you really want to
know in order to understand more about biology and biological
processes, is how the amounts of the proteins change in response to
different perturbations, like changes in the environment. Or if you
make a mutation in the cell, how did the abundance of the proteins
change,” said O'Shea.
The researchers' protein-localization studies revealed where some
4,156 proteins resided in the cell. According to Weissman, the
localization studies were performed at two levels of specificity.
“First, we examined the cells using microscopy techniques, and
for many proteins that was enough. We could tell that many proteins —
more than a thousand — existed in the nucleus or the cytoplasm.”
The nucleus contains the cell's genetic material and the cytoplasm is
the fluid-filled region outside the nucleus. “But for other
proteins, we saw a punctate pattern that only told us that the protein
was concentrated in a specific place.”
To pinpoint the locations of these proteins more specifically, the
researchers introduced genes for red fluorescent molecules that were
known to localize to one or another specific cell structure. Such
structures might include the mitochondria — the cell's power plant —
or the golgi apparatus, which is a network of internal cell membranes.
When the researchers saw both red and green fluorescence at a given
point, they knew that the protein concentrated in that structure.
According to O'Shea, the results of the localization studies were
gratifying, and are already beginning to have an impact. “We were
surprised that we could see as many proteins as we did, and that the
quality of the data was so good. Also, we were surprised that more than
eighteen hundred proteins have at least a part of their localization in
places other than the cytoplasm or the nucleus. So, from this study
we've gained a lot of information about potential new functions of
these proteins.”
O'Shea said that her future studies of localization, like those
quantifying protein levels, will concentrate on dynamic changes in the
cell. “In this study, we've only provided a static view of
localization under one condition,” she said. “But protein
localization is dynamic, in many cases, and I think that the big
challenge now is to use this library of strains to study how protein
localization changes in response to environmental
conditions.”
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