PLANT CLATHRIN HEAVY-CHAIN - SEQUENCE-ANALYSIS AND RESTRICTED LOCALIZATION IN GROWING POLLEN TUBES

Clathrin-coated vesicles were isolated from soybean (Glycine max L.) c ells in suspension culture and their purity was assessed using SDS-PAG E, peptide sequencing and electron microscopy. Antibodies raised to th ese coated vesicles were used to immunoscreen a soybean cDNA library i n lambda gt11 and isolate a partial clone of the clathrin heavy chain (HC) gene. Full-length cDNA for soybean clathrin HC was deduced by 5' and 3' cDNA amplification. The cDNA encodes an amino acid sequence of 1,700 residues, which is slightly larger than rat clathrin HC and may account for the reduced mobility of plant clathrin on SDS-PAGE. Insert ion of these extra residues is largely confined to the amino and carbo xy termini. Other domains within the heavy chain arms, including those implicated in light chain binding and trimerisation, are relatively w ell conserved between eukaryotes. A computer algorithm to determine al pha-helical coiled-coil structures reveals that only one domain, align ing to residues 1,450-1,489 in rat clathrin HC, has a high probability for coiled-coil structure in all five eukaryotic clathrin HC sequence s. This provides further evidence that the interaction between clathri n heavy and light chains is mediated by three bundles of coiled-coils near to the carboxy terminus. In analysing the role of plant clathrin in endocytotic trafficking, as against trafficking from the Golgi appa ratus to the vacuole, our attention was focused on membrane recyling i n tip-growing pollen tubes. These rapidly growing cells are highly sec retory and require a high level of plasma membrane recycling to mainta in the tube tip architecture. Monoclonal antibodies to plant clathrin HC confirmed that coated vesicles are relatively abundant in tip-growi ng pollen tubes of Lilium longiflorum. This analysis also demonstrated that a high proportion of the clathrin present is in an assembled sta te, suggesting a highly dynamic trafficking pathway. Immunofluorescenc e analysis of pollen tubes revealed that clathrin localises to the pla sma membrane at the apex of the pollen tube tip, which is consistent w ith high levels of clathrin-mediated membrane recycling. The use of th ese reagents in conjunction with tip-growing pollen tubes has created a unique opportunity to examine the basis for constitutive endocytosis , so that the more complex question of receptor-mediated pathways in p lants can also be assessed.