BACE is degraded via the lysosomal pathway

Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.     

Intracellular Itinerary of Internalised β‐Secretase,BACE1, and Its Potential Impact on β‐Amyloid Peptide Biogenesis

β‐Secretase (BACE1) cleavage of the amyloid precursor protein (APP) represents the initial step in the formation of the Alzheimer's disease associated amyloidogenic Aβ peptide. Substantive evidence indicates that APP processing by BACE1 is dependent on intracellular sorting of this enzyme. Nonetheless, knowledge of the intracellular trafficking pathway of internalised BACE1 remains in doubt. Here we show that cell surface BACE1 is rapidly internalised by the AP2/clathrin dependent pathway in transfected cells and traffics to early endosomes and Rab11‐positive, juxtanuclear recycling endosomes, with very little transported to the TGN as has been previously suggested. Moreover, BACE1 is predominantly localised to the early and recycling endosome compartments in different cell types, including neuronal cells. In contrast, the majority of internalised wild‐type APP traffics to late endosomes/lysosomes. To explore the relevance of the itinerary of BACE1 on APP processing, we generated a BACE1 chimera containing the cytoplasmic tail of TGN38 (BACE/TGN38), which cycles between the cell surface and TGN in an AP2‐dependent manner. Wild‐type BACE1 is less efficient in Aβ production than the BACE/TGN38 chimera, highlighting the relevance of the itinerary of BACE1 on APP processing. Overall the data suggests that internalised BACE1 and APP diverge at early endosomes and that Aβ biogenesis is regulated in part by the recycling itinerary of BACE1.     

Β-site APP-cleaving enzyme 1 trafficking and Alzheimer's disease pathogenesis

β-Site APP-cleaving enzyme (BACE1) cleaves the amyloid precursor protein (APP) at the β-secretase site to initiate the production of Aβ peptides. These accumulate to form toxic oligomers and the amyloid plaques associated with Alzheimer's disease (AD). An increase of BACE1 levels in the brain of AD patients has been mostly attributed to alterations of its intracellular trafficking. Golgi-associated adaptor proteins, GGA sort BACE1 for export to the endosomal compartment, which is the major cellular site of BACE1 activity. BACE1 undergoes recycling between endosome, trans-Golgi network (TGN), and the plasma membrane, from where it is endocytosed and either further recycled or retrieved to the endosome. Phosphorylation of Ser498 facilitates BACE1 recognition by GGA1 for retrieval to the endosome. Ubiquitination of BACE1 C-terminal Lys501 signals GGA3 for exporting BACE1 to the lysosome for degradation. In addition, the retromer mediates the retrograde transport of BACE1 from endosome to TGN. Decreased levels of GGA proteins and increased levels of retromer-associated sortilin have been associated with AD. Both would promote the co-localization of BACE1 and the amyloid precursor protein in the TGN and endosomes. Decreased levels of GGA3 also impair BACE1 degradation. Further understanding of BACE1 trafficking and its regulation may offer new therapeutic approaches for the treatment of Alzheimer's disease.     

Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the trans- Golgi Network (TGN) to lysosomes (t(1/2) approximately 30 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.     

Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes

Mutations in the mucolipin-1 gene have been linked to mucolipidosis type IV, a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. Mucolipin-1 is a membrane protein containing six putative transmembrane domains with both its N- and C-termini localized facing the cytosol. To gain information on the sorting motifs that mediate the trafficking of this protein to lysosomes, we have generated chimeras in which the N- and C- terminal tail portions of mucolipin-1 were fused to a reporter gene. In this article, we report the identification of two separate di-leucine-type motifs that co-operate to regulate the transport of mucolipin-1 to lysosomes. One di-leucine motif is positioned at the N-terminal cytosolic tail and mediates direct transport to lysosomes, whereas the other di-leucine motif is found at the C-terminal tail and functions as an adaptor protein 2-dependent internalization motif. We have also found that the C-terminal tail of mucolipin-1 is palmitoylated and that this modification might regulate the efficiency of endocytosis. Finally, the mutagenesis of both di-leucine motifs abrogated lysosomal accumulation and resulted in cell-surface redistribution of mucolipin-1. Taken together, these results reveal novel information regarding the motifs that regulate mucolipin-1 trafficking and suggest a role for palmitoylation in protein sorting.     

Amyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b‐ and AP4‐dependent pathway

The intracellular trafficking and proteolytic processing of the membrane‐bound amyloid precursor protein (APP) are coordinated events leading to the generation of pathogenic amyloid‐beta (Aβ) peptides. The membrane transport of newly synthesized APP from the Golgi to the endolysosomal system is not well defined, yet it is likely to be critical for regulating its processing by β‐secretase (BACE1) and γ‐secretase. Here, we show that the majority of newly synthesized APP is transported from the trans‐Golgi network (TGN) directly to early endosomes and then subsequently to the late endosomes/lysosomes with very little transported to the cell surface. We show that Arl5b, a small G protein localized to the TGN, and AP4 are essential for the post‐Golgi transport of APP to early endosomes. Arl5b is physically associated with AP4 and is required for the recruitment of AP4, but not AP1, to the TGN. Depletion of either Arl5b or AP4 results in the accumulation of APP, but not BACE1, in the Golgi, and an increase in APP processing and Aβ secretion. These findings demonstrate that APP is diverted from BACE1 at the TGN for direct transport to early endosomes and that the TGN represents a site for APP processing with the subsequent secretion of Aβ.      

Ubiquitination of the N-terminal Region of Caveolin-1 Regulates Endosomal Sorting by the VCP/p97 AAA-ATPase

Caveolin-1 (CAV1) is the defining constituent of caveolae at the plasma membrane of many mammalian cells. For turnover, CAV1 is ubiquitinated and sorted to late endosomes and lysosomes. Sorting of CAV1 requires the AAA+-type ATPase VCP and its cofactor UBXD1. However, it is unclear in which region CAV1 is ubiquitinated and how ubiquitination is linked to sorting of CAV1 by VCP-UBXD1. Here, we show through site-directed mutagenesis that ubiquitination of CAV1 occurs at any of the six lysine residues, 5, 26, 30, 39, 47, and 57, that are clustered in the N-terminal region but not at lysines in the oligomerization, intramembrane, or C-terminal domains. Mutation of Lys-5–57 to arginines prevented binding of the VCP-UBXD1 complex and, importantly, strongly reduced recruitment of VCP-UBXD1 to endocytic compartments. Moreover, the Lys-5–57Arg mutation specifically interfered with trafficking of CAV1 from early to late endosomes. Conversely and consistently, depletion of VCP or UBXD1 led to accumulation of ubiquitinated CAV1, suggesting that VCP acts downstream of ubiquitination and is required for transport of the ubiquitinated form of CAV1 to late endosomes. These results define the N-terminal region of CAV1 as the critical ubiquitin conjugation site and, together with previous data, demonstrate the significance of this ubiquitination for binding to the VCP-UBXD1 complex and for sorting into lysosomes.     

A Di-leucine signal in the ubiquitin moiety. Possible involvement in ubiquitination-mediated endocytosis

Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes upon ligand-dependent ubiquitination. However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial. In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins. The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently. Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis. We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu(43)-Ile(44). The Leu(43)-Ile(44) sequence mediated endocytosis and lysosomal sorting in a Leu(43)-dependent manner. These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.     

Differential use of two AP-3-mediated pathways by lysosomal membrane proteins

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.     

MARCH ubiquitin ligases alter the itinerary of clathrin-independent cargo from recycling to degradation

Following endocytosis, internalized plasma membrane proteins can be recycled back to the cell surface or trafficked to late endosomes/lysosomes for degradation. Here we report on the trafficking of multiple proteins that enter cells by clathrin-independent endocytosis (CIE) and determine that a set of proteins (CD44, CD98, and CD147) found primarily in recycling tubules largely failed to reach late endosomes in HeLa cells, whereas other CIE cargo proteins, including major histocompatibility complex class I protein (MHCI), trafficked to both early endosome antigen 1 (EEA1) and late endosomal compartments in addition to recycling tubules. Expression of the membrane-associated RING-CH 8 (MARCH8) E3 ubiquitin ligase completely shifted the trafficking of CD44 and CD98 proteins away from recycling tubules to EEA1 compartments and late endosomes, resulting in reduced surface levels. Cargo affected by MARCH expression, including CD44, CD98, and MHCI, still entered cells by CIE, suggesting that the routing of ubiquitinated cargo occurs after endocytosis. MARCH8 expression led to direct ubiquitination of CD98 and routing of CD98 to late endosomes/lysosomes.     

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.     

Intracellular trafficking of interleukin-1 receptor I requires Tollip

Interleukin-1 receptor (IL-1RI) is a master regulator of inflammation and innate immunity. When triggered by IL-1beta, IL-1RI aggregates with IL-1R-associated protein (IL-1RAcP) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. IL-1beta also rapidly triggers endocytosis of IL-1RI. Although internalization of IL-1RI significantly impacts signaling, very little is known about trafficking of IL-1RI and therefore about precisely how endocytosis modulates the overall cellular response to IL-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that IL-1beta induces ubiquitination of IL-1RI and that via these attached ubiquitin groups, IL-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of IL-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of IL-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding IL-1RI and ubiquitin), IL-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that IL-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of IL-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking IL-1RI, via Tom1, to the endosomal degradation machinery.     

An Endocytosed TGN38 Chimeric Protein Is Delivered to the TGN after Trafficking through the Endocytic Recycling Compartment in CHO Cells

To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and ¹²⁵I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38''s postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t _1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t _1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t _1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.     

Neuronal protein trafficking associated with Alzheimer disease

Aberrant and/or cumulative amyloid-beta (Aβ) production, resulting from proteolytic processing of the amyloid precursor protein (APP) by β and γ-secretases, have been postulated to be a main etiological basis of Alzheimer disease (AD). A number of proteins influence the subcellular trafficking itinerary of APP and the b-site APP-cleaving enzyme (BACE1) between the cell surface, endosomes and the trans-Golgi network (TGN). Available evidence suggests that co-residence of APP and BACE1 in the endosomal compartments promotes amyloidogenesis. Retrograde transport of APP out of the endosome to the TGN reduces Aβ production, while APP routed to and kept at the cell surface enhances its non-amyloidogenic, α-secretase-mediated processing. Changes in post-Golgi membrane trafficking in aging neurons that may influence APP processing is particularly relevant to late-onset, idiopathic AD. Dystrophic axons are key features of AD pathology, and impaired axonal transport could play crucial roles in the pathogenesis of idiopathic AD. Recent evidence has also indicated that Aβ-induced synaptic defects and memory impairment could be explained by a loss of both AMPA and NMDA receptors through endocytosis. Detail understanding of factors that influence these neuronal trafficking processes will open up novel therapeutic avenues for preventing or delaying the onset of symptomatic AD.     

Neuronal protein trafficking associated with Alzheimer disease: From APP and BACE1 to glutamate receptors

Aberrant and/or cumulative amyloid-beta (Aβ) production, resulting from proteolytic processing of the amyloid precursor protein (APP) by β and γ-secretases, have been postulated to be a main etiological basis of Alzheimer disease (AD). A number of proteins influence the subcellular trafficking itinerary of APP and the β-site APP-cleaving enzyme (BACE1) between the cell surface, endosomes and the trans-Golgi network (TGN). Available evidence suggests that co-residence of APP and BACE1 in the endosomal compartments promotes amyloidogenesis. Retrograde transport of APP out of the endosome to the TGN reduces Aβ production, while APP routed to and kept at the cell surface enhances its non-amyloidogenic, α-secretase-mediated processing. Changes in post-Golgi membrane trafficking in aging neurons that may influence APP processing is particularly relevant to late-onset, idiopathic AD. Dystrophic axons are key features of AD pathology, and impaired axonal transport could play crucial roles in the pathogenesis of idiopathic AD. Recent evidence has also indicated that Aβ-induced synaptic defects and memory impairment could be explained by a loss of both AMPA and NMDA receptors through endocytosis. Detail understanding of factors that influence these neuronal trafficking processes will open up novel therapeutic avenues for preventing or delaying the onset of symptomatic AD.Key words: amyloid precursor protein (APP), β-site APP cleaving enzyme 1 (BACE1), endosome, glutamate receptors, trans-Golgi network (TGN)     

Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments: evidence for early endosome-to-TGN trafficking of MPR300

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.     

A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking

The cation-independent mannose-6-phosphate receptor (CI-MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans-Golgi network (TGN) to lysosomes. Cycling of CI-MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS-1, which directs endosome-to-TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS-1 bind to an overlapping CI-MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2-activated phosphorylation cascade controlling PACS-1- and GGA3-mediated CI-MPR sorting. PACS-1 links GGA3 to CK2, forming a multimeric complex required for CI-MPR sorting. PACS-1-bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI-MPR and early endosomes. Bound CK2 also phosphorylates PACS-1Ser(278), promoting binding of PACS-1 to CI-MPR to retrieve the receptor to the TGN. Our results identify a CK2-controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system.     

Sorting of furin at the trans-Golgi network. Interaction of the cytoplasmic tail sorting signals with AP-1 Golgi-specific assembly proteins

The eukaryotic subtilisin-like endoprotease furin is found predominantly in the trans-Golgi network (TGN) and cycles between this compartment, the cell surface, and the endosomes. There is experimental evidence for endocytosis from the plasma membrane and transport from endosomes to the TGN, but direct exit from the TGN to endosomes via clathrin-coated vesicles has only been discussed but not directly shown so far. Here we present data showing that expression of furin promotes the first step of clathrin-coat assembly at the TGN, the recruitment of the Golgi-specific assembly protein AP-1 on Golgi membranes. Further, we report that furin indeed is present in isolated clathrin-coated vesicles. Packaging into clathrin-coated vesicles requires signal components in the furin cytoplasmic domain which can be recognized by AP-1 assembly proteins. We found that besides depending on the phosphorylation state of a casein kinase II site, interaction of the furin tail with AP-1 and its mu1subunit is mediated by a tyrosine motif and to less extent by a leucine-isoleucine signal, whereas a monophenylalanine motif is only involved in binding to the intact AP-1 complex. This study implies that high affinity interaction of AP-1 or mu1 with the cytoplasmic tail of furin needs a complex interplay of signal components rather than one distinct signal.     

SCF acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF^TrCP is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF^TrCP is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF^TrCP is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.