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Plant clathrin heavy chain: sequence analysis and restricted localisation in growing pollen tubes
H.D. Blackbourn, A.P. Jackson
Journal of Cell Science 1996 109: 777-786;

Summary

Clathrin-coated vesicles were isolated from soybean (Glycine max L.) cells in suspension culture and their purity was assessed using SDS-PAGE, peptide sequencing and electron microscopy. Antibodies raised to these coated vesicles were used to immunoscreen a soybean cDNA library in 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. Insertion of these extra residues is largely confined to the amino and carboxy termini. Other domains within the heavy chain arms, including those implicated in light chain binding and trimerisation, are relatively well conserved between eukaryotes. A computer algorithm to determine alpha-helical coiled-coil structures reveals that only one domain, aligning to residues 1,460-1,489 in rat clathrin HC, has a high probability for coiled-coil structure in all five eukaryotic clathrin HC sequences. This provides further evidence that the interaction between clathrin 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 apparatus to the vacuole, our attention was focused on membrane recycling in tip-growing pollen tubes. These rapidly growing cells are highly secretory and require a high level of plasma membrane recycling to maintain the tube tip architecture. Monoclonal antibodies to plant clathrin HC confirmed that coated vesicles are relatively abundant in tip-growing pollen tubes of Lilium longiflorum. This analysis also demonstrated that a high proportion of the clathrin present is in an assembled state, suggesting a highly dynamic trafficking pathway. Immunofluorescence analysis of pollen tubes revealed that clathrin localises to the plasma membrane at the apex of the pollen tube tip, which is consistent with high levels of clathrin-mediated membrane recycling. The use of these 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 plants can also be assessed.

REFERENCES

    1. Acton S. L.,
    2. Wong D. H.,
    3. Parham P.,
    4. Brodsky F. M. and
    5. Jackson A. P.
    (1993). Alteration of clathrin light chain expression by transfection and gene disruption. Mol. Biol. Cell 4, 647–660
    OpenUrlAbstract/FREE Full Text
    1. Bazinet C.,
    2. Katzen A. L.,
    3. Morgan M.,
    4. Mahowald A. P. and
    5. Lemmon S. K.
    (1993). The Drosophila clathrin heavy chain gene: Clathrin function is essential in a multicellular organism. Genetics 134, 1119–1134
    OpenUrlAbstract/FREE Full Text
    1. Brodsky F. M.
    (1988). Living with clathrin: Its role in intracellular membrane traffic. Science 242, 1396–1402
    OpenUrlAbstract/FREE Full Text
    1. Campbell C.,
    2. Squicciarini J.,
    3. Shia J.,
    4. Pilch P. F. and
    5. Fine R. E.
    (1984). Identification of protein kinase as an intrinsic component of rat liver coated vesicle. Biochemistry 23, 4420–4426
    OpenUrlCrossRefPubMed
    1. Cleveland D. W.
    (1983). Peptide mapping in one dimension by limited proteolysis of sodium dodecyl sulfate solubilized proteins. Meth. Enzymol 96, 222–229
    OpenUrlCrossRefPubMedWeb of Science
    1. Coleman J.,
    2. Evans D.,
    3. Hawes C.,
    4. Horsley D. and
    5. Cole L.
    (1987). Structure and molecular organisation of higher plant coated vesicles. J. Cell Sci 88, 35–45
    OpenUrlAbstract/FREE Full Text
    1. Galfre G. and
    2. Milstein C.
    (1981). Preparation of monoclonal antibodies: Stratagies and proceedures. Meth. Enzymol 73, 3–46
    OpenUrlCrossRefPubMed
    1. Goud B.,
    2. Huet C. and
    3. Louvard D.
    (1985). Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay. J. Cell Biol 100, 521–527
    OpenUrlAbstract/FREE Full Text
    1. Harley S. M. and
    2. Beevers L.
    (1989). Isolation and partial characterisation of clathrin coated vesicles from pea (Pisum sativum L.) cotyledons. Protoplasma 150, 103–109
    OpenUrlCrossRef
    1. Hoh B. and
    2. Robinson D. G.
    (1993). The prominent 28 kDa polypeptide inclathrin coated vesicle fractions from developing pea cotyledons is contaminating ferritin. Cell Biol. Int 17, 551–557
    OpenUrlCrossRef
    1. Jackson A. P. and
    2. Parham P.
    (1988). Structure of human clathrin light chains. J. Biol. Chem 263, 16688–16695
    OpenUrlAbstract/FREE Full Text
    1. Kirchhausen T.,
    2. Harrison S. C.,
    3. Chow E. P.,
    4. Mattaliano R. J.,
    5. Ramachandran K. L.,
    6. Smart J. and
    7. Brosius J.
    (1987). Clathrin heavy chain: molecular cloning and complete primary structure. Proc. Nat. Acad. Sci. USA 84, 8805–8809
    OpenUrlAbstract/FREE Full Text
    1. Knight A. E. and
    2. Kendrick-Jones J.
    (1993). A myosin-like protein from a higher plant. J. Mol. Biol 231, 148–154
    OpenUrlCrossRefPubMedWeb of Science
    1. Kohno T.,
    2. Chaen S. and
    3. Shimmen T.
    (1990). Characterisation of the translocator associated with pollen tube organelles. Protoplasma 154, 179–183
    OpenUrlCrossRefWeb of Science
    1. Laemmli U. K.
    (1970). Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227, 680–685
    OpenUrlCrossRefPubMedWeb of Science
    1. Lancelle S. A.,
    2. Cresti M. and
    3. Hepler P. K.
    (1987). Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes of Nicotiana alata. Protoplasma 140, 141–150
    OpenUrlCrossRefWeb of Science
    1. Lancelle S. A. and
    2. Hepler P. K.
    (1992). Ultrastructure of freeze-substituted pollen tubes of Lilium longiflorum. Protoplasma 167, 215–230
    OpenUrlCrossRefWeb of Science
    1. Lemmon S. K.,
    2. Pellicena-Palle A.,
    3. Conley K. and
    4. Freund C. L.
    (1991). Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function. J. Cell Biol 112, 65–80
    OpenUrlAbstract/FREE Full Text
    1. Lin H.-B.,
    2. Harley S. M.,
    3. Butler J. M. and
    4. Beevers L.
    (1992). Multiplicity of clathrin light chain-like polypeptides from developing pea (Pisum sativum L.) cotyledons. J. Cell Sci 103, 1127–1137
    OpenUrlAbstract/FREE Full Text
    1. Lupas A.,
    2. Dyke M. V. and
    3. Stock J.
    (1991). Predicting coiled coils from protein sequences. Science 252, 1162–1164
    OpenUrlFREE Full Text
    1. Maycox P. R.,
    2. Link E.,
    3. Reetz A.,
    4. Morris S. A. and
    5. Jahn R.
    (1992). Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling. J. Cell Biol 118, 1379–1388
    OpenUrlAbstract/FREE Full Text
    1. Mersey B. G.,
    2. Griffing L. R.,
    3. Rennie P. J. and
    4. Fowke L. C.
    (1985). The isolation of coated vesicles from protoplasts of soybean. Planta 163, 317–327
    OpenUrlCrossRefWeb of Science
    1. Mollenhauer H. H.,
    2. Morre D. J. and
    3. Griffing L. R.
    (1991). Post Golgi apparatus structures and membrane removal in plants. Protoplasma 162, 55–60
    OpenUrlCrossRefWeb of Science
    1. Nathke I. S.,
    2. Heuser J.,
    3. Lupas A.,
    4. Stockton J.,
    5. Turck C. W. and
    6. Brodsky F. M.
    (1992). Folding and trimerisation of clathrin subunits at the triskelion hub. Cell 68, 899–910
    OpenUrlCrossRefPubMedWeb of Science
    1. Noguchi T.
    (1990). Consumption of lipid granules and formation of vacuoles in the pollen tube of Tradescantia reflexa. Protoplasma 156, 19–28
    OpenUrlCrossRef
    1. O'Driscoll D.,
    2. Hann C.,
    3. Read S. M. and
    4. Steer M. W.
    (1993). Endocytotic uptake of fluorescent dextrans by pollen tubes grown in vitro. Protoplasma 175, 126–130
    OpenUrlCrossRefWeb of Science
    1. O'Halloran T. J. and
    2. Anderson R. G. W.
    (1992). Characterization of the clathrin heavy chain from Dictyostelium discoidium. DNA Cell Biol 11, 321–330
    OpenUrlCrossRefPubMedWeb of Science
    1. Ohno H.,
    2. Stewart J.,
    3. Fournier M.-C.,
    4. Bosshart H.,
    5. Rhee I.,
    6. Miyatake S.,
    7. Saito T.,
    8. Galluser A.,
    9. Kirchhausen T. and
    10. Bonifacino J. S.
    (1995). Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269, 1872–1875
    OpenUrlAbstract/FREE Full Text
    1. Pearse B. M. F. and
    2. Robinson M. S.
    (1990). Clathrin, adaptors and sorting. Annu. Rev. Cell Biol 6, 151–171
    OpenUrlCrossRefWeb of Science
    1. Picton J. M. and
    2. Steer M. W.
    (1981). Determination of secretory vesicle production rates by dictyosomes in pollen tubes of Tradescantia using cytochalasin D. J. Cell Sci 49, 261–272
    OpenUrlAbstract/FREE Full Text
    1. Robinson D. G. and
    2. Hillmer S.
    (1990). Endocytosis in plants. Physiol. Plant 79, 96–104
    OpenUrlCrossRef
    1. Sanger F.,
    2. Nicklen S. and
    3. Coulson A. R.
    (1977). DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. USA 74, 5463–5467
    OpenUrlAbstract/FREE Full Text
    1. Sorkin A. and
    2. Carpenter G.
    (1993). Interaction of activated EGF receptors with coated pit adaptins. Science 261, 612–615
    OpenUrlAbstract/FREE Full Text
    1. Tanchak M. A.,
    2. Griffing L. R.,
    3. Mersey B. G. and
    4. Fowke L.
    (1984). Endocytosis of cationised ferritin by coated vesicles of soybean protoplasts. Planta 162, 481–486
    OpenUrlCrossRefPubMedWeb of Science
    1. Van der Woude W. J.,
    2. Morre D. J. and
    3. Bracker C. E.
    (1971). Isolation and characterisation of secretory vesicles in germinated pollen of Lilium longiflorum. J. Cell Sci 8, 331–351
    OpenUrlAbstract/FREE Full Text
    1. Zhang J. Z.,
    2. Davletov B. A.,
    3. Sudhof T. C. and
    4. Anderson R. G. W.
    (1994). Synaptotagmin-1 is a high affinity receptor for clathrin AP2- Implications for membrane recycling. Cell 78, 751–760
    OpenUrlCrossRefPubMedWeb of Science
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Journal Articles
Plant clathrin heavy chain: sequence analysis and restricted localisation in growing pollen tubes
H.D. Blackbourn, A.P. Jackson
Journal of Cell Science 1996 109: 777-786;
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