matrix protein and yeast two-hybrid

Martin Hirst hirst at interomex.com
Wed Jan 15 18:05:01 EST 2003


Hi Franco,

Yes for the two-hybrid system to work at all the proteins must enter the
nucleus.  To increase the chance of this occurring two hybrid vectors (at
least the good ones ) have nuclear localization domains tagged to both the
bait and prey vector.  I guess the question would be what if the prey fusion
protein also contained an export signal sequence?  Which would dominant?

Cheers

Martin





On 1/14/03 10:05, "Vizeacoumar Joseph Franco" <francov at cellbnt.ualberta.ca>
wrote:

> Hi Martin,
> I have a question here,the whole idea of Yeast two hybrid takes place inside
> the nucleus because e of the DNA binding and activating domain. So whether
> it's a matrix or membrane protein they all enter the nucleus ( of course I
> agree as u said if it has many TM domains thens probably it gets stuck.) and
> normally there shouldn't be any problem right?
> Pls clarify.
> Rgds,
> Franco.
> 
> -----Original Message-----
> From: Martin Hirst [mailto:hirst at interomex.com]
> Sent: Monday, January 13, 2003 2:04 PM
> To: yeast at net.bio.net
> Subject: Re: matrix protein and yeast two-hybrid
> 
> I assume you mean all the interactions occur outside the cell?
> 
> One of the advantages of standard yeast two hybrid systems is that they
> level the playing field with respect to protein localization.  This can lead
> to false positives (extra-cellular/ intra-cellular etc.) but should not be a
> problem for you.  The problem of course is if the interaction you are
> looking for is dependent on a post-translational modification not applied to
> intracellular proteins (glycosylation etc...).
> 
> If your matrix protein contains a transmembrane domain I would suggest
> removing it as well.
> 
> There are other membrane based two hybrid systems out there which mount the
> bait protein onto the intracellular leaf of the cell membrane.  These
> systems work by triggering a signal transduction cascade when the bait/ prey
> interact.  Of course they are also internal so are bound by the same
> limitations as the standard transcription factor methods.
> 
> Cheers
> 
> Martin
> 
> 
>> Helo, I hope some people here in this forum familar with yeast
>> two-hybrid system. I am working on an extracellular protein and would
>> like to identify its interaction partners. I prefer yeast two-hybrid
>> approach but it is said that matrix molecule may not suit for the
>> screening since all the interactions happen inside the yeast cell.
>> However, several papers do describe the isiolation of the binding
>> proteins for matrix protein. Can anyone tell me what is chance to get
>> succeed? Or is there a modified two-hybrid system for matrix proteins?
>> Thanks!
>> 
>> 
> 



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