This web page was produced as an assignment for an undergraduate course at Davidson College

(Please see a more thorough assessment of sec18 from my homepage so that infornmation does not have to be
 repeated)

    The interactions that a protein undergoes are essential to its function.  Signal transduction pathways involving
hormones such as glucagon and epinephrine are just one example of the relationship between function and
protein-protein interaction.  To better understand the role that sec18 plays in the life of yeast, one can also analyze
the proteins that interact with sec18.  Eisenburgs database <http://dip.doe-mbi.ucla.edu/dip/Search_DIP.cgi>, for
example, predicts that sec18 interacts with PEP12.  Such a prediction is validated by the literature on sec18.
PEP12 is a t-Snare protein that mediates fusion between vesicles from the golgi and the vacuole.  sec18, moreover,
has been shown to mediate the interaction of t-Snare and v-Snare proteins between vesicles from the vacuole,
golgi, and the ER.  Thus it is a reasonable conclusion drawn by Eisenburgs database that sec18 would interact with
PEP12.
    Stan Fields database at Washington University <http://depts.washington.edu/%7Eyeastrc/th_11.htm> used a
yeast-two hybrid system to predict proteins that would interact with sec18.  His results showed that BET3, in
addition to PEP12, interacts with sec18.  The interaction between sec18 and BET3 is also consistent with the
literature.  BET3 acts in concert with SNARE proteins to mediate the targeting and fusion of ER vesicles with the
golgi.  Because sec18 acts in a similar fashion, these data raise the possibility that sec18 and BET3 may form a
dimer to mediate membrane fusion.  Such information is crucial for future experiments as some traditional
techniques rely on breaking non-covalent interactions between proteins to study their function, thus failing to mimic
the true conditions under which that protein may operate.  With experiments that take into account the natural
affinity between sec18 and BET3 a more accurate representation of the proteome as a web is drawn out,
suppressing the false view that each protein acts in isolation.
    The Yeast Proteome Database <http://www.proteome.com/databases/YPD/YPDsearch-quick.html> does not cite
the method it employs to list proteins that interact with sec18 but it does mention that sec18 interacts with LMA1.
Like the other interactions already noted involving sec18, this interaction is consistent with the literature.  It has
been noted that sec18 interacts with LMA1 so that it may hydrolyze ATP, resulting in its dissociation from the
complex of the two vesicles.  Only after sec18 dissociates from the two vesicles can those vesicles fuse as the
pretein needed for the final stage of fusion (Ypt7p) only acts upon the dissolution of the V/T Snare complex.
    Although the aforementioned mechanism is supported by experimental evidence there are others who hold this
mechanism in low regard.  The putative interaction between sec18 and LMA1 has more than a few skeptics.  If it is
assumed that the hydrolysis of ATP provides the energy for sec18 to dissociate from the pre-fusion complex
between the vesicles before they fuse, then one can apply a subtle variation of the two-hybrid system to support the
notion that LMA1 interacts with sec18 so that sec18 may hydrolyze ATP.  Using sec18 as bait and LMA1 as prey
in a two-hybrid system, one could incubate the system with radioactive ATP and use biochemical methods to
quantify the release of radioactive Pi.  If the interaction between sec18 and LMA1 does lead to the hydrolysis of
ATP, then a yeast-two hybrid system set up in this way should yield more radioactive Pi than a control experiment,
which would involve measuring the quantity of Pi in a system involving only bait (sec18) or prey (LMA1) but not
both in the same system.
    In my previous analysis with the microarray expression of sec18, it was shown that sec18 is repressed during
certain parts of the cell cycle.  I proposed that one reason for this may be that vesicular fusion could, during these
parts of the cell cycle, adversely affect the cell.  Although repression is one way to ensure that a protein does not
carry out its normal function, another method the cell employs to regulate the activities of a protein is
phosphorylation.  To date, no studies have been performed that assess the possibility that sec18 may be
phosphorylated in the cell.  To do so, one could take protein samples from yeast in response to different
environmental conditions or at different points in the cell cycle, perform 2-D protein electrophoresis, and use mass
spectrometry to analyze the mass/charge ratios for peptides corresponding to sec18.  If sec18 is phosphorylated, its
mass/charge ratio will change and these data will show those conditions under which sec18 could be
phosphorylated.  If sec18 is modified in this way, we can make predictions as to how phosphorylation affects the
activity of this protein and under what conditions sec18 may be most active.
    The many interactions that sec18 partakes in with other proteins make the cell seem much more like a web with
connected parts than a simple collection of different parts.  As the cell web grows, researchers are trying to find
proteins with similar structures to other proteins to aid in deciphering functional similarities and the cell web in
particular.  Here is an overlay of two proteins both known to participate in vesicular fusion.  They are very similar
in function and may provide the groundwork for characterizing motifs consistent with proteins involved in vesicular
fusion: (This figure was obtained from The Dali Server at <http://www2.ebi.ac.uk/dali/>  Permission is being sought to keep the image here.)
 


 


References Cited

1) Database of Interacting Proteins [Online Database].  Available from: <http://dip.doe-mbi.ucla.edu/dip/Search_DIP.cgi>  Accessed 2001 Nov 8

2) Yeast Resource Center [Online Database].  Available from:<http://depts.washington.edu/%7Eyeastrc/th_home.htm> Accessed 2001 Nov 8

3) Yeast Protein Database [Online Database]. Available from: <http://www.proteome.com/databases/YPD/YPDsearch-quick.html> Accessed 2001 Nov 8

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