Scientists Determine Identity, Cell Locale And Quantity Of Nearly All Proteins In An Organism

Science Daily | University Of California - San Francisco ^ | 2003-10-16

[sourcery]

UCSF scientists have developed a set of powerful tools that allow researchers to look in unprecedented detail at the full complement of thousands of proteins acting and interacting in a living organism. They have used the new tools to mine nearly the entire proteome of an organism ? discovering what proteins are active in each cell, where they are active and in what quantity.

The results, published in two papers in the October 16 issue of the journal Nature, provide the most comprehensive and detailed picture yet of protein activity in the living cells of higher organisms. They reveal a tremendous range in the amount of different proteins active in a cell and help explain the "logic" of the timing and regulation of gene activity.

Recognizing that it is the gene's products -- the proteins ? that interact to regulate all life processes, many researchers have begun to turn their attention from the roster of all the genes in an organism - the genome -- to the proteome. But while new technologies such as gene chips and whole genome sequencing have accelerated studies of the genome, the technology to study the whole proteome has lagged far behind.

Unlike genes, which all use the same code, each protein has unique physical properties, thwarting attempts to develop a single method to study them all in a living cell. By giving each protein a common tag, the UCSF scientists were able to follow where each protein acted and in what quantity.

With this new method for a thorough protein search, they were able to examine thousands of proteins individually -- one in each of thousands of different strains of the common baker's yeast, Saccharomyces cerevisiae, an organism that has proven to be a good model for understanding basic biology of human cells. About one third of the yeast's genes are shared by humans, and each yeast cell contains a nucleus and other basic organelles that function in humans cells.

By scrutinizing only one protein in each strain under study, the researchers determined precisely where and in what quantity each protein acts during normal growth -- a tool that can now be used to identify the role of different proteins, the relationship between sets of genes and proteins, and the links between protein changes and disease.

The new approach was far more sensitive than any previous attempt to detect low levels of protein activity, the scientists reported. They detected proteins in quantities as low as 50 molecules per cell ? an extraordinarily small amount to detect -- and as high as a million or more molecules per cell, demonstrating that living cells utilize different proteins in vastly different amounts.

By integrating the new protein findings with those of gene studies, the scientists were able to decipher some of the logic by which certain genes are normally turned on and off together. They found that such "co-regulated" genes code for proteins that reside in the same place in the cell.

"Things make more sense when you can look at the level of proteins ? the actual players in the life of cells and organisms," said Jonathan Weissman, PhD, a Howard Hughes Medical Institute (HHMI) investigator, professor of cellular and molecular pharmacology at UCSF, and co-senior author on both papers.

The gene messages are "imperfect proxies" for the information we really care about, which is the proteins," Weissman added. "The reason we've been looking at the messages ? the genes -- is because we could, but what is more informative is proteins ? where they act, how they act and in what kind of abundance."

"There has been a real lack of good tools to follow what proteins were doing in a cell in any comprehensive way. Because of this, fundamental questions remained unanswerable about the proteome and its relationship to the genome. Now, we can address those questions."

Altogether, 4,200 proteins were identified -about 80 percent of all the different proteins in the baker's yeast.

Erin O'Shea, PhD, also an HHMI investigator and UCSF professor of biochemistry and biophysics, is also co-senior author on both papers.

The scientists used two identical sets of thousands of strains of the yeast -- one to determine where in the cell each protein acts, and the other to determine the abundance of each protein. In the study of each protein's cellular locale, they "tagged" a different gene in each yeast strain.

The tag was a green florescent marker, attached to the gene in such a way that it was not likely to disrupt the gene's function. When the gene carried out its normal role, instructing the cell to make a specific protein, that protein glowed green, and could be located in the living cell using microscopy. Since each yeast strain had only one tagged protein, the researchers could pinpoint the location of nearly all of the organism's proteins by looking for the telltale green sign in each strain.

At the outset, they assumed that yeast contain about 6,200 proteins, since other scientists had identified that many genes in DNA, but through this and a second tagging approach, the researchers found that only about 5,500 of the suspected genes actually coded for proteins.

They used the second collection of yeast strains to tag the proteins with a standard marker that allows purification and measurement of the abundance of the protein. When the results of the two parallel approaches were pooled, the team was able to identify the type, cell location and abundance of about 80 percent of the yeast's proteins.

The scientists were not able to study all of the intended 5,500 proteins. The study could only identify a protein if its genes expressed it - if the protein was active. Yeast have at least two major phases in their life cycle: the main period of feeding, growth and reproduction, and at least one phase in which the organism responds to specialized stress conditions and produces spores - sort of like abandoning ship. The scientists expect that a significant proportion of the proteins they did not identify simply are not expressed in the normal growth phase that they studied.

In addition, some proteins may be expressed in such small amounts - less than 50 molecules per cell, for example - that they are below the detection level of even this highly sensitive approach. Finally, for strictly technical reasons, some of the proteins may have escaped successful tagging.

Nonetheless, the project was easily thorough enough to net some striking findings. For example, the research revealed that of the 4,200 yeast proteins scrutinized, a full 527 of them -- or about one in eight - work in the energy-converting organelle, the mitochondrion. O'Shea speculates that the mitochondrion may have such a large share of the proteins because, in addition to it vital energy conversion function, this organelle is believed to be derived from a separate organism - a bacterial parasite or pathogen that invaded our ancient ancestral cell. The mitochondrion, as scientists now believe, is a direct descendent of an independent organism that simply "moved in" and started keeping house with its host.

Weissman and O'Shea have known each other since graduate school in 1989. Both were named Howard Hughes Medical Institute investigators at UCSF in 2000 -prestigious and generous support for biomedical scientists.

"HHMI urged all the recipients to do something particularly challenging," O'Shea says. "So, Jonathan and I decided to pool our HHMI resources to try to develop the tools need to study proteomics."

"Genomics was led by technology that we didn't have before," Weissman says. "Proteomics can't advance unless we develop new technology to study thousands of proteins in a systematic way. That's what we set out to do."

The research was completed at the Center for Advanced Technology at UCSF's new Mission Bay campus. The CAT is intended to support development of new technologies.

Lead authors on the Nature article, "Global Analysis of Protein Localization in Budding Yeast" are Won-Ki Huh, PhD and James Falvo, PhD, postdoctoral fellows in O'Shea's lab. Co-authors are Luke C. Gerke, BS, research associate; Adam S. Carroll, PhD, research specialist; and Russell W. Howson, graduate student.

Lead author on the Nature letter in the same issue, "Global Analysis of Protein Expression in Budding Yeast," is Sina Ghaemmaghami, PhD, postdoctoral fellow in Weissman's lab. Co-authors are Won-Ki Huh; Kiowa Bower, BA, research technician; Russell Howson; Archana Belle, PhD, postdoctoral fellow; and graduate student Noah Dephoure.

In addition to the Howard Hughes Medical Institute, the research was supported by the David and Lucille Packard Foundation.

[gore3000]

But your objection was that eucaryotes, or their ancestors, could not have acquired mitochondria at some point in time becuase they would have needed them to produce energy to begin with. That objection was not valid because many cells can and do produce energy without mitochondria (or chloroplast, or any other specialized organelles).

It certainly is valid. A mitochondria or a chloroplast is an organ, a part of an organism. As I said, no one is stupid enough to say that stomachs and lungs were parasitic organisms. Clearly these organelles were not parasitic organisms either since they are not organisms in any way, they just do a very specialized function which is totally useless by itself.

[general_re]

As 0.5 mm is absolutely huge when it comes to bacteria, one can pretty much dismiss this "expert's" claims.

He probably means "micrometers" rather than "millimeters", which would be the correct size for M. pneumoniae - it is one of the smallest bacteria. Sloppy work, though.

[Stultis]

Of course the theory is that the ancestors of mitochondria were free living organisms which first entered other cells as parasites, and that over time an obligate relationship developed with mitochondria developing their specialized character. So, your first objection, that the ancestors of eucaryotes could not have survived without mitochondria is false, and now your second objection, that the mitochondria could not have survived because they were too specialized, turns out to be a strawman. Would you like to try for three?

[Junior]

Don't you use a special symbol for micrometers to differentiate it from millimeters? Something like µ? I wouldn't have expected the original poster to catch such a glaring error.

[general_re]

That's how I always did it - micrometers = µm. I assume that's what the intention was, since a quick lookup says that the size range for that bacterium is 0.2 µm to 0.8 µm, and that it is one of the smallest known bacteria. That's what I assume, but I don't really know - maybe he really meant "millimeters". Either way, it's wrong on some level ;)

[webstersII]

bump for later read.

[Right Wing Professor]

Don't you use a special symbol for micrometers to differentiate it from millimeters?

Yep, you use the greek letter mu. In symbol font, that's m; I suspect Gore3000 didn't notice the font change when he pasted.

[js1138]

Fonts are a bit tricky in html. When I cut and past anything more than just text, I first paste it into a good html editor that generates browser nonspecific code. I then paste the code into FR. Sometimes the appearance changes a bit, but original intent is still clear.

[Junior]

Hold down your ALT key and type 0181 on your number pad. That way you don't need to change fonts.

[js1138]

My html editor -- homesite -- has a clickable "keyboard" with all the available characters. Fat chance I'm going to memorize all the codes for non-typewriter characters.

[Junior]

I don't memorize them (actually I do memorize a few —, £, ×, •). I use the character map function in Windows.

[gore3000]

Of course the theory is that the ancestors of mitochondria were free living organisms which first entered other cells as parasites, and that over time an obligate relationship developed with mitochondria developing their specialized character. So, your first objection, that the ancestors of eucaryotes could not have survived without mitochondria is false, and now your second objection, that the mitochondria could not have survived because they were too specialized, turns out to be a strawman. Would you like to try for three?

I never said that 'eucaryotes could not have survived without mitochondria'. You are putting words in my mouth I never said. What I said is that no living thing can exist without the complex system that makes energy in the form of ATP in living things. There are numerous systems that do this from the bacterial systems, to the chloroplasts and mitochondria in eucaryotes. So eucaryotes could not have survived a single day without a system to make ATP from day one. The claim that mitochondria came to give eucaryotes what they needed is therefore false. Further, we have the problem here of 'the dog ate the homework' which we see in so much evolutionary rhetoric. There is no sign of eucaryotes ever having had anything but mitochondria (or chloroplasts in plants) to fulfill this function. So this is another example of evolutionists making a story out of whole cloth or what I like to call 'fact free science'.

As to the part about mitochondria not being able to survive without the cell, that is not a strawman. Just because they have their own DNA does not mean that they can survive on their own. Remember what I said from the start - no living thing can survive without a system to produce enerby. While mitocondria are part of the energy production system in cells, they are not the whole:

The food we eat must first be converted to basic chemicals that the cell can use. Some of the best energy supplying foods contain sugars or carbohydrates ...bread, for example. Using this as an example, the sugars are broken down by enzymes that split them into the simplest form of sugar which is called glucose. Then, glucose enters the cell by special molecules in the membrane called “glucose transporters”.

Once inside the cell, glucose is broken down to make ATP in two pathways. The first pathway requires no oxygen and is called anaerobic metabolism. This pathway is called glycolysis and it occurs in the cytoplasm outside the mitochondria. During glycolysis, glucose is broken down into pyruvate. Other foods like fats can also be broken down for use as fuel (see following cartoon). Each reaction is designed to produce some hydrogen ions (electrons) that can be used to make energy packets (ATP). However, only 4 ATP molecules can be made by one molecule of glucose run through this pathway. That is why mitochondria and oxygen are so important. We need to continue the breakdown process with the Kreb’s cycle inside the mitochondria in order to get enough ATP to run all the cell functions.
From: Mitochondrial Substructure.

So much evo 'fact free science' (or should I call it pseudo-science?).

[Stultis]

So, your third argument boils down to eucaryotes weren't eucaryotes before they were eucaryotes. Well, congradulations. You can't argue with that!

[gore3000]

So, your third argument boils down to eucaryotes weren't eucaryotes before they were eucaryotes. Well, congradulations. You can't argue with that!

Nope, my argument, my sole argument is and has been from the start that mitochondria are and always were an organ, an integral part of animal eucaryotes. Neither you, nor phony evo scientists have shown any evidence otherwise - including the writers of this fantasy article.

[Stultis]

my sole argument is and has been from the start that mitochondria are and always were

Yes, I see. Your sole argument is bald assertion / sheer denial. Thanks for the clarification!

an organ, an integral part of animal eucaryotes. Neither you, nor phony evo scientists have shown any evidence otherwise

And here we have a strawman. Of course the relationship between eucaryotes and their mitchondria or chloroplasts has become obligate (in most cases, anyway; in some protists the chloroplasts can be destroyed without harming the organism). No one denies this. But there is plenty of evidence for the endosymbiotic theory (that these organelles were originally symbiotic bacteria), e.g.:

• Both mitochondria and chloroplasts can arise only from preexisting mitochondria and chloroplasts. They cannot be formed in a cell that lacks them because nuclear genes encode only some of the proteins of which they are made.
• Both mitochondria and chloroplasts have their own genome and it resembles that of prokaryotes not that of the nuclear genome.
  • Both genomes consist of a single circular molecule of DNA.
  • There are no histones associated with the DNA.
• Both mitochondria and chloroplasts have their own protein-synthesizing machinery, and it more closely resembles that of prokaryotes than that found in the cytoplasm of eukaryotes.
  • The first amino acid of their transcripts is always fMet as it is in bacteria (not methionine [Met] that is the first amino acid in eukaryotic proteins).
  • A number of antibiotics (e.g., streptomycin) that act by blocking protein synthesis in bacteria also block protein synthesis within mitochondria and chloroplasts. They do not interfere with protein synthesis in the cytoplasm of the eukaryotes.
  • Conversely, inhibitors (e.g., diphtheria toxin) of protein synthesis by eukaryotic ribosomes do not — sensibly enough — have any effect on bacterial protein synthesis nor on protein synthesis within mitochondria and chloroplasts.
  • The antibiotic rifampicin, which inhibits the RNA polymerase of bacteria, also inhibits the RNA polymerase within mitochondria. It has no such effect on the RNA polymerase within the eukaryotic nucleus.

source

[exDemMom]

Don't you use a special symbol for micrometers to differentiate it from millimeters? Something like µ? I wouldn't have expected the original poster to catch such a glaring error.

When you cut and paste, the "µ" symbol often becomes an "m", since it is really the same letter, using the "Symbol" font instead of the "Times New Roman" font. This was a huge problem for me when I was a beginning graduate student (eons ago). I'd see "0.5 mL" in a recipe, and I didn't have the experience to know it was really "0.5 µL" and, for some reason, the reaction wouldn't work as it was supposed to. Luckily, I know better now.

[exDemMom]

The tag was a green florescent marker, attached to the gene in such a way that it was not likely to disrupt the gene's function. When the gene carried out its normal role, instructing the cell to make a specific protein, that protein glowed green, and could be located in the living cell using microscopy. Since each yeast strain had only one tagged protein, the researchers could pinpoint the location of nearly all of the organism's proteins by looking for the telltale green sign in each strain.

At the outset, they assumed that yeast contain about 6,200 proteins, since other scientists had identified that many genes in DNA, but through this and a second tagging approach, the researchers found that only about 5,500 of the suspected genes actually coded for proteins.

I am amazed. Unless one is actively working in the field, I doubt anyone can appreciate the magnitude of this work. And to tag each protein not just once, but again in a second strain of yeast... mind-boggling. I wonder how long it took, how many researchers were involved in the project, and exactly what approach they took in tagging each protein. (Of course, if I just go get the papers I could answer a couple of these questions right away.)

[sourcery]

Unless one is actively working in the field, I doubt anyone can appreciate the magnitude of this work.

Yes. And many probably wouldn't realize the impact such technology will have on science.

[gore3000]

Yes, I see. Your sole argument is bald assertion / sheer denial. Thanks for the clarification!

You keep lying about my postings even though they are just above, pretty shameless.

But there is plenty of evidence for the endosymbiotic theory (that these organelles were originally symbiotic bacteria)

Both mitochondria and chloroplasts can arise only from preexisting mitochondria and chloroplasts.

This is false. Since all eukaryotic organisms have one or the other and this is an essential function without the cell cannot survive they are an integral part of the eukaryotic cell.

Both mitochondria and chloroplasts have their own genome and it resembles that of prokaryotes not that of the nuclear genome.

Indeed they do but these genomes are very small and cannot and could never have provided the essentials of life for any individual organism.

The other similarities again do not speak to the problem that these could never have been individual organisms. They lack not only the essentials for a living organism, but they even lack - as I already pointed out in post# 32 - the essential proteins and materials for the production of ATP - their sole reason of being - and again an essential of any living thing.

So what we have here is again what I have been stating - fact free science otherwise known as Barbara Streisand or artbellian science. The only reason for this totally factualless assumption by atheist/evolutionists is that the theory of evolution requires it. The facts against it are quite strong:
1. the chloroplasts and mitochondria do not have the means of ever having been free living organisms.
2. the chloroplasts and mitochondria do not have the means of even producing the essentials of their function - ATP without proteins from the nuclear organism of a eukaryote.
3. neither chloroplasts or mitochondria have ever been seen as free living organisms.
4. both chloroplasts and mitochondria perform an essential function in the organisms in which they are found. These functions are highly integrated into the organisms in which they are found. There is no trace of the replacement of the essential function of ATP production in any of the organisms in which they are found.
5. There is also no trace of the reduction of the chloroplast and mitochondrial genomes which would have been necessary if these had ever been free living organisms.

In short there is no evidence that these chloroplasts and mitochondria were ever anything but what they are now. Therefore my statement is correct, this is just another story made up out of whole cloth by evolutionists to justify their theory in absence of any facts to support it.

[PatrickHenry]

PatrickHenry

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