I am replying to your bionet request for ideas about purification of
Chlamydomonas plasma membrane. This is certainly a long standing problem
that has attracted the attention of a lot of people but has not seen much
success, as far as I can see. The basic problem is that the cell body plasma
membrane of Chlamydomonas is sandwiched tightly between and probably
connected to both the glycoprotein cell wall and the enormous chloroplast.
The only feasible approach that I see is to use a really clean cell wall-less
mutant (which one is best?; many leave cell wall layers/components on the
cell surface) or utilize lysin plus cell wall synthesis inhibitors to get rid
of and keep off the wall (but does one really get a totally naked plasma
membrane? or for how long) and then to biotinylate the surface of the cells.
One can get rid of the flagellar membrane by deflagellation either before
cell wall removal and biotinylation or afterwards. Then one has to break up
the cells somehow (gently would be nice but one has to be rough enough to
physically dissociate cytoplasmic organelles and membrane contamination such
as fragments of the chloroplast) in the presence of lots of protease
inhibitors, and then use some sort of affinity chromatography on
avidin-sepharose beads to pull out the fragments/vesicles of biotinylated
plasma membrane. One could couple this approach with continuous sucrose
density centrifugation after cell disruption and before avidin-sepharose
chromatography. Or one could stick with the sucrose density purification and
use the biotinylation as a method of assessing purity of the preparation by
doing TEM analysis using gold-conjugated avidin. I have not actually pursued
this combination of approaches or made many attempts to purify plasma
membrane as my interest is in the flagellar membrane but there are a number
of us (certainly Bloodgood, Snell, Goodenough, van den Ende) who would like
to compare the composition of the flagellar membrane to the cell body plasma
membrane (some of us in vegetative cells; others in gametic cells). I know
the biotinylation of Chlamydomonas cell surfaces components is dead easy (bad
choice of terms) and very (and I mean very) gentle on the cells - we do it
all the time and would be glad to provide the details upon request (there
aren't really many details necessary). Hope these ideas may be a bit helpful
and may stimulate some vigorous give and take on the bionet Chlamy newsgroup
- we need some increased level of discussion on the Chlamy newsgroup. If you
get a bunch of private responses, I hope you will consolidate them and put
them on the Chlamy newsgroup for everyone's benefit.
I understand that Charlene Forest has been working on purification of
Chlamy plasma membrane and she told me something about a phase partition
method she has been using. I would suggest asking her for details (Charlene,
are you on the bionet?). Charlene is at Brooklyn College Biology Department
- phone 718-780-5709; FAX 718-859-8383.
Bob Bloodgood, University of Virginia
RAB4m at Virginia.edu
