Peptide Motifs: Building the Clathrin Machinery

Abstract

Clathrin-coated vesicles (CCVs) form at the plasma membrane, where they select cargo for endocytic entry into cells, and at the trans-Golgi network (TGN) and the endosomal system, where they generate carrier vesicles that transport proteins between these compartments. We have used subcellular fractionation and tandem mass spectrometry to identify proteins associated with brain CCVs. The resulting proteome contained a near complete inventory of the major functional proteins of synaptic vesicles (SVs), suggesting that clathrin-mediated endocytosis provides a major mechanism to recycle SV membrane proteins following neurotransmitter release. Additionally, we identified several new components of the machineries for clathrin-mediated membrane budding, including enthoprotin/epsinR and NECAP 1/2. These proteins bind with high specificity to the ear domains of the clathrin adaptor proteins (APs)-1 and -2, and, intriguingly, they each utilize novel peptide motifs based around the core sequence ØXXØ. Detailed mutational analysis of these motifs, coupled with structural studies of the ear domains, has revealed the basis of their specificity for clathrin adaptors. Moreover, the motifs have now been recognized in multiple proteins functioning in clathrin-mediated membrane trafficking, revealing new mechanisms in the formation and function of CCVs. Thus, proteomics analysis of isolated organelles can provide insights ranging from peptide motifs to global organelle function.

Connecdenn, A Novel DENN Domain-Containing Protein of Neuronal Clathrin-Coated Vesicles Functioning in Synaptic Vesicle Endocytosis

“…In nerve terminals, AP-2 in a complex with stonin 2 interacts with phosphatidylinositol(4,5)P 2 and synaptotagmin, allowing for coats to nucleate on SV membranes after SV fusion with the plasma membrane (Haucke and De Camilli, 1999;Diril et al, 2006). AP-2 also serves as a scaffolding platform for a battery of endocytic accessory proteins, including epsin, huntingtin interacting protein 1, AP180, amphiphysin, disabled 2, autosomal recessive hypercholesteremia gene product, and NECAP 1 (Slepnev and De Camilli, 2000;McPherson and Ritter, 2005;Traub, 2005). A common feature of these proteins is the presence of N-terminal globular domains, including E/ANTH (epsin/AP180 N-terminal homology) domains and BAR domains, which function in generating and/or sensing membrane curvature, and PTB (phosphotyrosine binding) domains, which serve as binding modules for endocytic cargo proteins (Traub, 2003;Legendre-Guillemin et al, 2004;McMahon and Gallop, 2005).…”

Zebrafish as a Genetic Model in Biological and Behavioral Gerontology: Where Development Meets Aging in Vertebrates – A Mini-Review

“…and Scottish subjects [42] and in two Latin-American populations [43] , though not in a Japanese population [44] . The role of enthoprotin in regulating the transport and stability of clathrin-coated vesicles, and its enrichment in brain, suggests that it might be important for recycling of synaptic vesicles [45][46][47] . Thus, impairment in the function of this protein might be associated with degraded cognitive performance.…”

The AP-2 Adaptor β2 Appendage Scaffolds Alternate Cargo Endocytosis

“…YXXØ-type sorting signals (as found in the transferrin receptor) are bound directly by the 2 subunit (Ohno et al, 1995), whereas [DE]XXXL [LIM] dileucine signals are recognized by a functional hemicomplex of the ␣ and 2 subunit (Janvier et al, 2003;Coleman et al, 2005;Chaudhuri et al, 2007). Various assembly protein and clathrin associations are established through the ␣ and ␤2 appendages (Wang et al, 1995;McPherson and Ritter, 2005;Traub, 2005; This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -07-0712) on October 8, 2008. 2006b; Schmid and McMahon, 2007). The ␣ appendage has two interaction surfaces, one each upon the platform and sandwich subdomains.…”

“…YXXØ-type sorting signals (as found in the transferrin receptor) are bound directly by the 2 subunit (Ohno et al, 1995), whereas [DE]XXXL [LIM] dileucine signals are recognized by a functional hemicomplex of the ␣ and 2 subunit (Janvier et al, 2003;Coleman et al, 2005;Chaudhuri et al, 2007). Various assembly protein and clathrin associations are established through the ␣ and ␤2 appendages (Wang et al, 1995;McPherson and Ritter, 2005 Abbreviations used: ARH, autosomal recessive hypercholesterolemia protein; CLASP, clathrin-associated sorting protein; Dab2, disabled-2; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; HC, heavy chain; LDL, low-density lipoprotein; mAb, monoclonal antibody; MPR, cation-independent mannose 6-phosphate receptor; PtdIns(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; PTB, phosphotyrosine binding; tdRFP, tandem dimer tomato; Tf488, transferrin conjugated to Alexa Fluor 488; Tf568, transferrin conjugated to Alexa Fluor 568; Tf633, transferrin conjugated to Alexa Fluor 633; YFP, yellow fluorescent protein.© 2008 by The American Society for Cell Biology 53092006b; Schmid and McMahon, 2007). The ␣ appendage has two interaction surfaces, one each upon the platform and sandwich subdomains.…”