The control of the synthesis, assembly and segregation of distinct surface domains in a model unicellular system will be addressed in this proposed study. In Euglena a surface fraction and its subfractions enriched in plasma membranes and membrane skeletons will be independently evaluated and compared with a flagellar fraction that lacks or differs in these components. First surface isolates will be used to resolve the dynamics of attachment of the membrane skeleton to the plasma membrane by calculating turnover rates of the membrane skeletal proteins and by detailing the behavior of unassembled cellular pools of these proteins. Secondly, to what extent the membrane skeletal proteins themselves determine surface organization will be evaluated in experiments in which proteins with slightly different biochemical properties but markedly different assembly patterns are separately or in combination reassociated on plasma membranes. In a third set of experiments the significance of a relatively minor membrane skeletal protein that shares immunological determinants with spectrin will be assessed by peptide mapping, overlay experiments and cloning. Fourthly, in order to gain a fuller understanding of the timing of synthesis and assembly of replicating surfaces and regenerating flagella, molecular probes will be constructed for specific integral and peripheral membrane proteins. Recombinant from a cDNA library in the expression vector, Agt11 have yielded clones tentatively identified as coding for cell surface-specific proteins, a flagellar specific protein as well as a and B tubulins. Clones will be further confirmed, recloned and restriction mapped. Sequencing of the cDNA coding for an integral membrane protein will be given first priority as this protein appears to be a putative membrane skeletal anchor restricted to the cell surface. It is anticipated that DNA probes derived from sequences specific for proteins of the cell surface or the flagellar surface will be useful ultimately for isolating genomic clones and examining upstream regions for sequences common to genes coding for proteins targeted to either of those two domains. The results of these experiments will significantly increase our understanding of how membrane domains are maintained, and how cell surface components interact with components of the cellular interior.
