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

1996 ◽  
Vol 109 (4) ◽  
pp. 777-786 ◽  
Author(s):  
H.D. Blackbourn ◽  
A.P. Jackson
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Coiled Coil ◽  
Pollen Tubes ◽  
Peptide Sequencing ◽  
Coated Vesicles ◽  
Membrane Recycling ◽  
Clathrin Heavy Chain ◽  
Partial Clone ◽  
Sds Page

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.

Structure of clathrin cages and coats in ice

1986 ◽  
Vol 44 ◽  
pp. 94-97
Author(s):  
G.P.A. Vigers ◽  
R.A. Crowther ◽  
B.M.F. Pearse
Keyword(s):  
Electron Microscopy ◽  
Heavy Chain ◽  
Outer Part ◽  
Coated Vesicles ◽  
Eukaryotic Cells ◽  
Coat Proteins ◽  
Terminal Domain ◽  
Clathrin Heavy Chain ◽  
Membrane Components

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.

Structure and molecular organization of higher plant coated vesicles

1987 ◽  
Vol 88 (1) ◽  
pp. 35-45
Author(s):  
JULIAN COLEMAN ◽  
DAVID EVANS ◽  
CHRIS HAWES ◽  
DAVID HORSLEY ◽  
LOUISE COLE
Keyword(s):  
Heavy Chain ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Coated Vesicles ◽  
Molecular Organization ◽  
Higher Plant ◽  
Thin Sectioning ◽  
Sds Page ◽  
Protease Treatment ◽  
Dry Cleaving

Suspension-cultured cells of carrot contain three populations of coated vesicles, associated with the plasma membrane (84–91 nm diameter), Golgi dictyosomes and the partially coated reticulum (61–73 nm diameter). These were observed by thin sectioning, dry-cleaving and rapid-freeze deep-etching of cells. Dissociation of clathrin coats with Tris, released triskelions that were morphologically identical with those from mammalian tissue. The triskelion arm length of carrot clathrin was greater (61nm versus 44–50 nm), but packaging results in clathrin cages of pentagons and hexagons of similar size to those from mammalian cells. SDS-PAGE of Tris-released triskelion preparations revealed a complex of three polypeptides of 190, 60 and 57(x103)Mr. The 190x103Mr protein is the plant clathrin heavy chain, slightly larger than the mammalian heavy chain. The 60 and 57(x103)Mr bands showed the same sensitivities to protease treatment as mammalian light chains. Triskelion preparations containing these three proteins reassembled into polyhedral cages. These results are discussed in relation to the structural organization of coated vesicles and clathrin cages in other systems.

scholarly journals The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

1996 ◽  
Vol 7 (11) ◽  
pp. 1667-1677 ◽  
Author(s):  
K Redding ◽  
M Seeger ◽  
G S Payne ◽  
R S Fuller
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Heavy Chain ◽  
Intracellular Transport ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Localization Signal ◽  
Mutant Forms ◽  
Kex2 Protease

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.

scholarly journals Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function.

1991 ◽  
Vol 112 (1) ◽  
pp. 65-80 ◽  
Author(s):  
S K Lemmon ◽  
A Pellicena-Palle ◽  
K Conley ◽  
C L Freund
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Heavy Chain ◽  
Coiled Coil ◽  
Coated Vesicle ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Clathrin Heavy Chain ◽  
And Function ◽  
Major Defect

The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.

scholarly journals The structures of natively assembled clathrin-coated vesicles

2020 ◽  
Vol 6 (30) ◽  
pp. eaba8397 ◽  
Author(s):  
Mohammadreza Paraan ◽  
Joshua Mendez ◽  
Savanna Sharum ◽  
Danielle Kurtin ◽  
Huan He ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Associated Factors ◽  
Structural Model ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Clathrin Heavy Chain ◽  
Assembled Complexes ◽  
The Individual

Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

scholarly journals Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Gilles Moulay ◽  
Jeanne Lainé ◽  
Mégane Lemaître ◽  
Masayuki Nakamori ◽  
Ichizo Nishino ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Genetic Control ◽  
Heavy Chain ◽  
Muscle Development ◽  
Tissue Homeostasis ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Coated Pits ◽  
Clathrin Heavy Chain

Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene (CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance.

scholarly journals Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

1999 ◽  
Vol 10 (8) ◽  
pp. 2687-2702 ◽  
Author(s):  
Francesc Tebar ◽  
Stefan K. Bohlander ◽  
Alexander Sorkin
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Myeloid Leukemia ◽  
Protein Localization ◽  
Adaptor Protein ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Clathrin Heavy Chain ◽  
Golgi Network ◽  
The Impact

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure–function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP–CALM was targeted to the plasma membrane–coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP–CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP–CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin–CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Molecular Forms of Human Protein C: Comparison and Distribution in Human Adult Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 902-905 ◽  
Author(s):  
Brian S Greffe ◽  
Marilyn J Manco-Johnson ◽  
Richard A Marlar
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Single Chain ◽  
Beta Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Adult Plasma ◽  
The Difference

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

Immunolocalization of H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

PROTOPLASMA ◽  
1992 ◽  
Vol 171 (1-2) ◽  
pp. 55-63 ◽  
Author(s):  
G. Obermeyer ◽  
M. L�tzelschwab ◽  
H. -G. Heumann ◽  
M. H. Weisenseel
Keyword(s):  
Plasma Membrane ◽  
Pollen Grains ◽  
Pollen Tubes
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