Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

Abstract

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin-and to a lesser extent the other AP complexes-are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

“…Each AP complex recognizes a subset of sorting motifs, and the variant amino acids within [DERQ]XXXL [LI] and YXXΦ sequences define the specificity of signal recognition (47)(48)(49). Disruption of AP complex interactions with sorting motifs can lead to missorting of cargo proteins back to the plasma membrane (50)(51)(52)(53). The AP2 complex recognizes proteins with more divergent dileucine motifs and binds tyrosine motifs with higher affinity than AP1 and AP3, a property thought to promote retrieval of proteins missorted to the plasma membrane (54,55).…”

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

“…The siRNA were purchased from Dharmacon (Lafayette, CO) as nucleotide duplexes with 3′dTdT overhangs, designed to target human γ1 (5′-GGAAGAGCCUAUUCAGGUA-3′), γ2 (5′-AAACCCUGCUUUGC-UGUUAA-3′) (Rost et al, 2006) and μ1A (5′-GGCAUCAAGUAUCGG-AAGA-3′) (Janvier and Bonifacino, 2005). An alternative siRNA targeting γ2 (γ2 siRNA#2; 5′-CACAGAACACAGCAUAUCU-3′), with a 3′dTdT overhang, was purchased from Bioneer Inc (Alameda, CA).…”

Canonical Interaction of Cyclin G–associated Kinase with Adaptor Protein 1 Regulates Lysosomal Enzyme Sorting

“…RNA interference (RNAi) in HeLa cells was carried out as described previously (Janvier and Bonifacino, 2005). Small interfering RNA (siRNA) for CHC was also described previously (Janvier and Bonifacino, 2005).…”

“…Small interfering RNA (siRNA) for CHC was also described previously (Janvier and Bonifacino, 2005). SmartPool siRNA for human AP1-␥1 and a custom-designed siRNA for human GAK (5Ј-AACGAAGGAACAGCUGAUUCA-3Ј) were purchased from Dharmacon, (Chicago, IL).…”