Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: The cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier
Hunnicutt,Gary R.; Kosfiszer,M.G.; Snell,William J.
Journal of Cell Biology 111(4): 1605-1616
Publication date: 1990
Fertilization in Chlamydomonas reinhardtii is initiated when gametes ofopposite mating types adhere to each other via adhesion molecules(agglutinins) on their flagella. Adhesion leads to loss of activeagglutinins from the flagella and recruitment of new agglutinins from apool associated with the cell body. We have been interested in determiningthe precise cellular location of the pool and learning more about therelationship between agglutinins in the two domains. In the studiesreported here we describe methods for purification of mt+ cell bodyagglutinins by use of ammonium sulfate precipitation, chromatography(molecular sieve, ion exchange, and hydrophobic interaction), and sucrosegradient centrifugation. About 90% of the total agglutinins were associatedwith the cell body and the remainder were on the flagella. Cell bodyagglutinins were indistinguishable from mt+ flagellar agglutinins bySDS-PAGE, elution properties on a hydrophobic interaction column, and insedimentation properties on sucrose gradients. The nonadhesiveness of cellbodies suggested that the cell body agglutinins would be intracellular, butour results are not consistent with this interpretation. We havedemonstrated that brief trypsin treatment of deflagellated gametesdestroyed all of the cell body agglutinins and, in addition, we showed thatthe cell body agglutinins were accessible to surface iodination. Theseresults indicated that C. reinhardtii agglutinins have a novel cellulardisposition: active agglutinins, representing approximately 10% of thetotal cellular agglutinins, are found only on the flagella, whereas theremaining 90% of these molecules are on the external surface of the cellbody plasma membrane in a nonfunctional form. This segregation of celladhesion molecules into distinct membrane domains before gameticinteractions has been demonstrated in sperm of multicellular organisms andmay be a common mechanism for sequestering these critical molecules untilgametes are activated for fusion. In experiments in which surface-iodinated cell bodies were permitted to regenerate new flagella, we foundthat the agglutinins (as well as the 350,000 Mr, major flagellar membraneprotein) on the newly regenerated flagella were iodinated. These resultsindicate that proteins destined for the flagella can reside on the externalsurface of the cell body plasma membrane and are recruited onto newlyforming flagella as well as onto preexisting flagella during fertilization.