Recognition of sorting signals by clathrin adaptors

Heilker, Ralf; Spiess, Martin; Crottet, Pascal

doi:10.1002/(sici)1521-1878(199907)21:7<558::aid-bies4>3.0.co;2-r

Cited by 126 publications

(112 citation statements)

References 85 publications

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“…To investigate this possibility, we co-stained for g-adaptin (Figure 7a), which is a subunit of the clathrin-associated protein (AP-1) complex that, in turn, is associated with vesicle budding at the TGN. 26,27 We observed a steadily increasing percentage of total PB overlapping with g-adaptin for up to 2 h of uptake, after which the percentage of overlapping PB decreased to nearly negligible levels (Figure 7b). …”

Section: Pb Avoids Lysosomes and Coincides With Alternative Pathway Mmentioning

confidence: 89%

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

et al. 2006

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The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptormediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to a v -integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through nonclathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrinand non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells. Gene Therapy (2006) 13, 821-836.

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Section: Pb Avoids Lysosomes and Coincides With Alternative Pathway Mmentioning

confidence: 89%

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

et al. 2006

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“…It is of interest to note that the penton proteins in the Ad capsid contain a consensus endosomal sorting motif, the dileucine (LL) motif. Proteins that interact with the LL motif include clathrin-associated adapter proteins, 42,43 effectors required for normal secretory vesicle maturation in some specialized secretory cells. 44 Findings that the HIV Nef protein utilizes this same motif to recruit clathrin-associated adapters suggest that this motif in other viral proteins is functional.…”

Section: Discussionmentioning

confidence: 99%

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

et al. 2004

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Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in 480% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UVinactivated Ad, carbachol-stimulated release of bulk protein and b-hexosaminidase were significantly (Pp0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.

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“…TfR and other constitutively endocytosing proteins such as the low-density lipoprotein (LDL) are internalized due to their constitutive binding to the AP-2 complex, which mediates their recruitment into clathrin-coated pits. These internalization motifs consist of either a dileucine motif or an essential tyrosine residue in the context of NPXY or YXXØ, where X is any amino acid and Ø is a bulky, hydrophobic amino acid (Heilker et al, 1999). Disruption of this interaction by mutations in either the constitutively endocytosing cargo or in AP-2 abrogates TfR internalization (Nesterov et al, 1999).…”

Section: The Journey To the Lysosome: Major Highlightsmentioning

confidence: 99%

Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases

2004

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Recognition of sorting signals by clathrin adaptors

Cited by 126 publications

References 85 publications

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases

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