Polymerization of clathrin protomers into basket structures

Jaarsveld, Van; Pk, Nandi; Re, Lippoldt; Saroff, H. A.; Edelhoch, Harold

doi:10.1021/bi00517a028

Cited by 47 publications

(47 citation statements)

References 22 publications

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“…However, clathrin lattice assembly under optimal conditions is known to proceed extremely rapidly (Van Jaarsveld et al, 1981), raising the possibility that the anomalous flat lattices observed may have formed only during or subsequent to shearing of the upper membrane and breakage.…”

Section: Discussionmentioning

confidence: 99%

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

Santini

Marks

Keen

1998

MBoC

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The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals. INTRODUCTIONThe initial steps in the formation of clathrin-coated pits (CPs) at the plasma membrane and trans-Golgi network (TGN) involve the recruitment to the membrane of clathrin and either AP2 (at the plasma membrane) or AP1 (in the TGN). Substantial cytosolic pools of these proteins exist in cells, often equaling or exceeding the membrane-bound forms (Goud et al., 1985). Several studies have shown that hormonal stimulation can lead to rapid and sustained several-fold increases in plasma membrane CPs under certain conditions, consistent with mobilization of these soluble pools (Connolly et al., 1981(Connolly et al., , 1984Fisher and Rebhun, 1983). Nonetheless, little is known about the details of CP formation at the plasma membrane.The finding that AP2 binds to specific internalization signals (endocytic motifs) in the cytoplasmic domains of certain transmembrane receptors (Pearse, 1988;Glickman et al., 1989) led to the speculation that the receptors themselves may initiate CP assembly by recruiting AP2 to the membrane (Pearse and ‡ Corresponding author. 1 Abbreviations used: CP, Coated pit; M6P, mannose-6-phosphate; TGN, trans-Golgi network.© 1998 by The American Society...

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Section: Discussionmentioning

confidence: 99%

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

Santini

Marks

Keen

1998

MBoC

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“…However, there are major differences: the clathrin cages from various sources contain mainly three proteins, of molecular masses 180, 36, and 33 kDa; they have a larger diameter, of 65-125 nm (13); and the cages formed in vitro have an S value of about 300 (18). Also in contrast to clathrin cages (14), exposure to low pH (6.0) or high pH (8.5) did not dissociate the S-complex aggregates.…”

Section: Discussionmentioning

confidence: 99%

Secretory S complex of Bacillus subtilis forms a large, organized structure when released from ribosomes.

Caulfield¹,

Tai²,

Davis³

1985

Proc. Natl. Acad. Sci. U.S.A.

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The S complex of Bacillus subtilis, a set of four proteins that appears to be involved in protein secretion, is shown to be attached to 70S ribosomes: antibody to its 64-kDa component can aggregate these ribosomes, and the complex can be chemically crosslinked to ribosomal proteins. Low Mg2+ or prolonged high-speed centrifugation in a sucrose gradient releases the S complex from the ribosomes, and it is recovered as an aggregate with an S value of 76. Electron microscopy shows that these aggregates have a regular structure, somewhat resembling clathrin cages, with a diameter of about 45 nm. If these aggregates are physiological, their function would differ significantly from that of the signal recognition particle of eukaryotes.A 64-kDa protein of Bacillus subtilis, present in the membrane fraction complexed with ribosomes (complexed membrane, CM), appears to be involved in protein secretion since the ribosomes protect it from interaction with protease or antibody (1); it is also present in the cytosol (2, 3). In addition, this protein is present in a complex of four proteins (the S complex), of 64, 60, 41, and 36 kDa, found in large amounts in the membrane-free monosomes (but not in the membrane-polysome complexes or in the cytosol) (3). These findings suggest that the S complex functions in the initiation step in protein secretion, like the signal recognition particle of eukaryote cells (4), but it differs significantly: it lacks an RNA molecule (at least as isolated), and it failed to cause translational arrest at the signal sequence of a secretory protein (3). The distribution of the 64-kDa protein further suggests that in the initiation cycle the other proteins of the S complex may be released after contact with the membrane, while the 64-kDa protein remains attached and then is released at a later stage. This paper shows that the S complex separates from the ribosomes when they are dissociated into subunits by low Mg2+ or by high-speed centrifugation. However, the complex then sediments at about 76 S, rather than at the expected low values. Since the S complex in the monosome fraction can be chemically crosslinked to certain ribosomal proteins and anti-64-kDa IgG can aggregate the monosomes (as shown by electron microscopy), the 76S particle is evidently an aggregate of S complex, formed after release from attachment to the ribosomes. Moreover, after release, but not in the monosome fraction, the S complex is seen in the electron microscope as a specific, organized structure, larger than the ribosome. It is intriguing that this structure somewhat resembles the clathrin cages of eukaryotic cells, which have been implicated in membrane traffic (5 buffer. To decrease contamination with 50S subunits and disomes the preparation was passed through a second 10-30% sucrose gradient and the 70S region was recovered. In another method for preparing monosomes associated with S complex, the initial supernate from the lysate, after the bulk ofthe membrane/ribosomes had been removed by a 1-hr centrifugation (3),...

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“…6 c contain a high proportion of microcages, and correspondingly, nerve cells are well-known to acidify postmortally. In any case, our current feeling is that microcage formation will turn out to be a good indication of cytoplasmic acidification, and probably occurs via mechanisms similar to in vitro polymerization of clathrin by dialysis into acidic solutions (23,38,44,47). This is discussed in more detail in the accompanying paper (16).…”

Section: The Formation Of Microcagesmentioning

confidence: 97%

Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.

Heuser

Anderson

1989

The Journal of Cell Biology

879

707

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Abstract. Two seemingly unrelated experimental treatments inhibit receptor mediated endocytosis: (a) depletion of intracellular K ÷ (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson. 1983. Cell. 33:273-285); and (b) treatment with hypertonic media (Daukas, G., and S. H. Zigmond. 1985. J. Cell Biol. 101:1673-1679. Since the former inhibits the formation of clathrin-coated pits (Larkin, J. M., W. D. Donzell, and R. G. W. Anderson. 1986. J. Cell Biol. 103:2619-2627, we were interested in determining whether hypertonic treatment has the same effect, and if so, why. Fibroblasts (human or chicken) were incubated in normal saline made hypertonic with 0.45 M sucrose, then broken open by sonication and freezeetched to generate replicas of their inner membrane surfaces. Whereas untreated cells display typical geodesic lattices of clathrin under each coated pit, hypertonic cells display in addition a number of empty clathrin "microcages'. At first, these appear around the edges of normal coated pit lattices. With further time in hypertonic medium, however, normal lattices largely disappear and are replaced by accumulations of microcages. Concomitantly, low density lipoprotein (LDL) receptors lose their normal clustered distribution and become dispersed all over the cell surface, as seen by fluorescence microscopy and freeze-etch electron microscopy of LDL attached to the cell surface. Upon return to normal medium at 37°C, these changes promptly reverse. Within 2 min, small clusters of LDL reappear on the surfaces of cells and normal clathrin lattices begin to reappear inside; the size and number of these receptor/clathrin complexes returns to normal over the next 10 min. Thus, in spite of their seeming unrelatedness, both K ÷ depletion and hypertonic treatment cause coated pits to disappear, and both induce abnormal clathrin polymerization into empty microcages. This suggests that in both cases, an abnormal formation of microcages inhibits endocytosis by rendering clathrin unavailable for assembly into normal coated pits. C LATHRIN coated pits are the primary plasma membrane specialization involved in the uptake of a wide variety of molecules by receptor-mediated endocytosis (4, 38). Two broad functions have been attributed to these regions of membrane: (a) molecular determinants associated with the clathrin lattice may cause receptors to become clustered; and (b) the clathrin lattice may in some way control the invagination of the membrane to form endocytic vesicles. To understand the molecular mechanisms underlying these two aspects of coated pit function, one approach is to search for treatments that inhibit endocytosis and then to characterize the effects of these treatments on coated pit structure and receptor clustering.Currently, three experimental treatments are known to inhibit receptor-mediated endocytosis: (a) depletion of intracellular potassium (22, 24-26, 29, 30, 34, 40), (b) exposure of cells to hypertonic media (10), and (c) acidification of the cytoplasm (11,16, 21,41). Whereas potassi...

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Polymerization of clathrin protomers into basket structures

Cited by 47 publications

References 22 publications

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

Secretory S complex of Bacillus subtilis forms a large, organized structure when released from ribosomes.

Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.

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