Plant clathrin heavy chain: sequence analysis and restricted localisation in growing pollen tubes

Area of Science:

• Molecular Biology
• Plant Cell Biology
• Biochemistry

Background:

• Clathrin-coated vesicles are essential for intracellular trafficking in eukaryotes.
• Understanding plant clathrin heavy chain (HC) structure and function is key to elucidating endocytosis in plants.
• Tip-growing pollen tubes exhibit high secretory activity, necessitating efficient membrane recycling.

Purpose of the Study:

• To isolate and characterize clathrin-coated vesicles from soybean (Glycine max L.) cells.
• To identify and analyze the soybean clathrin heavy chain (HC) gene and its encoded protein.
• To investigate the role of clathrin in membrane recycling within tip-growing pollen tubes.

Main Methods:

• Isolation and purity assessment of clathrin-coated vesicles using SDS-PAGE, peptide sequencing, and electron microscopy.
• Immunoscreening of a soybean cDNA library to isolate clathrin HC gene fragments.
• 5' and 3' cDNA amplification to obtain the full-length clathrin HC cDNA sequence.
• Bioinformatic analysis of the deduced amino acid sequence, including coiled-coil structure prediction.
• Immunofluorescence microscopy to localize clathrin in Lilium longiflorum pollen tubes.

Main Results:

• A partial and then full-length cDNA for soybean clathrin HC was isolated, encoding a 1,700-residue protein.
• The plant clathrin HC sequence shows conserved domains for light chain binding and trimerization, with variations primarily at the termini.
• A specific coiled-coil domain (residues 1,460–1,489 in rat HC) is conserved across eukaryotes, suggesting a conserved interaction mechanism.
• Coated vesicles are abundant in tip-growing pollen tubes, with clathrin predominantly in an assembled state.
• Clathrin localizes to the plasma membrane at the apex of pollen tube tips, supporting its role in membrane recycling.

Conclusions:

• Soybean clathrin HC shares conserved structural features with other eukaryotes, particularly in domains mediating protein interactions.
• The findings provide evidence for clathrin-mediated endocytosis and membrane recycling in plant pollen tubes.
• This study establishes a foundation for investigating receptor-mediated endocytosis in plants using pollen tube models.