Lysosomal targeting of P-selectin is mediated by a novel sequence within its cytoplasmic tail

Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.     

Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 --> Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 --> Ala or Phe13 --> Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.     

Children''s emerging regulation of conduct: restraint, compliance, and internalization from infancy to the second year

Receptor-mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.     

Fluid-phase marker transport in rat liver: free-flow electrophoresis separates distinct endosome subpopulations

Free-flow electrophoresis (FFE) was used to investigate the intracellular compartments involved in fluid-phase marker, fluoresceine isothiocyanate (FITC)-dextran, transport in the isolated perfused rat liver. One to 2 min after uptake at 37 degrees C, FITC-dextran was found in endosomes with the same electrophoretic mobility as early sorting endosomes labeled either by the hepatocyte-specific marker asialoorosomucoid (ASOR) or by transferrin that enters all liver cells. Labeling at low temperature (16 degrees C) blocked transport of ASOR and dextran in early endosomes. With increasing internalization time (3-13 min) at 37 degrees C, FITC-dextran-labeled compartments co-localized with late, ASOR-containing endosomes. Since localization of FITC-dextran in late transcytotic compartments was not observed upon FFE separation, it is concluded that the majority of internalized markers is directed to lysosomes. The FITC-label did not account for the predominant lysosomal targeting of the dextran, since [3H]dextran-labeled endosomes exhibited an identical FFE pattern. Taken together, these data indicate that the fluid-phase marker dextran is transported through intracellular compartments with identical characteristics as endosome subcompartments of the receptor-mediated lysosomal route.     

Lumenal and transmembrane domains play a role in sorting type I membrane proteins on endocytic pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.     

MHC class II transport from lysosomal compartments to the cell surface is determined by stable peptide binding, but not by the cytosolic domains of the alpha- and beta-chains

Inside APCs, MHC class II molecules associate with antigenic peptides before reaching the cell surface. This association takes place in compartments of the endocytic pathway, more related to endosomes or lysosomes depending on the cell type. Here, we compared MHC class II transport from endosomal vs lysosomal compartments to the plasma membrane. We show that transport of MHC class II molecules to the cell surface does not depend on the cytosolic domains of the alpha- and beta-chains. In contrast, the stability of the alphabeta-peptide complexes determined the efficiency of transport to the cell surface from lysosomal, but not from endosomal, compartments. In murine B lymphoma cells, SDS-unstable and -stable complexes were transported to the cell surface at almost similar rates, whereas after lysosomal relocalization or in a cell line in which MHC class II molecules normally accumulate in lysosomal compartments, stable complexes were preferentially addressed to the cell surface. Our results suggest that when peptide loading occurs in lysosomal compartments, selective retention and lysosomal degradation of unstable dimers result in the expression of highly stable MHC class II-peptide complexes at the APC surface.     

Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.     

Herpes simplex virus gD and virions accumulate in endosomes by mannose 6-phosphate-dependent and -independent mechanisms

Herpes simplex virus (HSV) glycoprotein D (gD) is modified with mannose 6-phosphate (M6P) and binds to M6P receptors (MPRs). MPRs are involved in the well-characterized pathway by which lysosomal enzymes are directed to lysosomes via a network of endosomal membranes. Based on the impaired ability of HSV to form plaques under conditions in which glycoproteins could not interact with MPRs, we proposed that MPRs may function during HSV egress or cell-to-cell spread (C. R. Brunetti, R. L. Burke, B. Hoflack, T. Ludwig, K. S. Dingwell, and D. C. Johnson, J. Virol. 69:3517-3528, 1995). To further analyze M6P modification and intracellular trafficking of gD in the absence of other HSV proteins, adenovirus (Ad) vectors were used to express soluble and membrane-anchored forms of gD. Both membrane-bound and soluble gD were modified with M6P residues and were localized to endosomes that contained the 275-kDa MPR or the transferrin receptor. Similar results were observed in HSV-infected cells. Cell fractionation experiments showed that gD was not present in lysosomes. However, a mutant form of gD and another HSV glycoprotein, gI, that were not modified with M6P were also found in endosomes in HSV-infected cells. Moreover, a substantial fraction of the HSV nucleocapsid protein VP6 was found in endosomes, consistent with accumulation of virions in an endosomal compartment. Therefore, it appears that HSV glycoproteins and virions are directed to endosomes, by M6P-dependent as well as by M6P-independent mechanisms, either as part of the virus egress pathway or by endocytosis from the cell surface.     

A determinant in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor prevents trafficking to lysosomes

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function.     

Structural requirements for major histocompatibility complex class II invariant chain endocytosis and lysosomal targeting

The invariant chain (Ii) targets newly synthesized major histocompatibility complex class II complexes to a lysosome-like compartment. Previously, we demonstrated that both the cytoplasmic tail (CT) and transmembrane (TM) domains of Ii were sufficient for this targeting and that the CT contains two di-leucine signals, 3DQRDLI8 and 12EQLPML17 (Odorizzi, C. G., Trowbridge, I. S., Xue, L., Hopkins, C. R., Davis, C. D., and Collawn, J. F. (1994) J. Cell Biol. 126, 317-330). In the present study, we examined the relationship between signals required for endocytosis and those required for lysosomal targeting by analyzing Ii-transferrin receptor chimeras in quantitative transport assays. Analysis of the Ii CT signals indicates that although 3DQRDLI8 is necessary and sufficient for endocytosis, either di-leucine signal is sufficient for lysosomal targeting. Deletions between the two signals reduced endocytosis without affecting lysosomal targeting. Transplantation of the DQRDLI sequence in place of the EQLPML signal produced a chimera that trafficked normally, suggesting that this di-leucine sequence coded for an independent structural motif. Structure-function analysis of the Ii TM region showed that when Ii TM residues 11-19 and 20-29 were individually substituted for the corresponding regions in the wild-type transferrin receptor, lysosomal targeting was dramatically enhanced, whereas endocytosis remained unchanged. Our results therefore demonstrate that the structural requirements for Ii endocytosis and lysosomal targeting are different.     

Free-flow electrophoretic analysis of endosome subpopulations of rat hepatocytes

The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells. Here, using 125I-labeled anti-secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 degrees C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.     

Studies of small GTPase Rab7 association with endosomes of cells expressing normal and mutant forms of epidermal growth factor receptors

A study was made of an association of small GTPase Rab7, commonly considered as a marker of late endosomes, with endosomal compartments of cells expressing EGF receptor with different ability to be sorted for degradative pathway. It was found that in cells HER14, expressing normal EGF receptor, Rab7 was associated with both early and late endosomes and the extent of association correlated with the number of EGF-receptor complexes in the specific endosomal fraction. Cels with a receptor, lacking major sites of autophosphorylation by deletion of 126 C-terminal residues (CD126), demonstrated a low efficiency of EGF-receptor sorting to late endosomes and decreased association dynamics with Rab7. Interaction of Rab7 with endosomes of cells expressing kinase negative receptors (K721) was found to be minimal. At the same time, in cells Cd126 and K721 with a low sorting efficiency Rab7 was mainly associated with early endosomes. These data favor Rab7 involvement in mediating early-to-late endosomal transition.     

Effect of helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation

The effect of vacuolating toxin (VacA) from Helicobacter pylori on endosomal and lysosomal functions was studied by following procathepsin D maturation and epidermal growth factor (EGF) degradation in HeLa cells exposed to the toxin. VacA inhibited the conversion of procathepsin D (53 kDa) into both the intermediate (47 kDa) and the mature (31 kDa) form. Nonprocessed cathepsin D was partly retained inside cells and partly secreted in the extracellular medium via the constitutive secretion pathway. Intracellular degradation of EGF was also inhibited by VacA with a similar dose-response curve. VacA did not alter endocytosis, cell surface recycling, and retrograde transport from plasma membrane to trans-Golgi network and endoplasmic reticulum, as estimated by using transferrin, diphtheria toxin, and ricin as tracers. Subcellular fractionation of intoxicated cells showed that procathepsin D and nondegraded EGF accumulate in lysosomes. Measurements of intracellular acidification with fluorescein isothiocyanate-dextran revealed a partial neutralization of the lumen of endosomes and lysosomes, sufficient to account for both mistargeting of procathepsin D outside the cell and the decreased activity of lysosomal proteases.     

Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter

We have studied the involvement of proteolytic pathways in the regulation of the Na/Pi cotransporter type II by parathyroid hormone (PTH) in opossum kidney cells. Inhibition of lysosomal degradation (by leupeptin, ammonium chloride, methylamine, chloroquine, L-methionine methyl ester) prevented the PTH-mediated degradation of the transporter, whereas inhibition of the proteasomal pathway (by lactacystin) did not. Moreover it was found (i) that whereas lysosomal inhibitors prevented the PTH-mediated degradation of the transporter they did not prevent the PTH-mediated inhibition of the Na/Pi cotransport and (ii) that treating opossum kidney cells with lysosomal inhibitors led to an increased expression of the transporter without any concomitant increase in the Na/Pi cotransport. Further analysis by subcellular fractionation and morphological techniques showed (i) that the Na/Pi cotransporter is constitutively transported to and degraded within late endosomes/lysosomes and (ii) that PTH leads to the increased degradation of the transporter in late endosomes/lysosomes.     

Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes

In this study we take advantage of recently developed methods using J774 macrophages to prepare enriched fractions of early endosomes, late endosomes, dense lysosomes, as well as phagosomes of different ages enclosing 1-micron latex beads to investigate the steady state distribution and trafficking of lysosomal enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to phagolysosomes, where acid hydrolases were concentrated. The first site of hydrolase concentration was the early endosomes, which contained the bulk of the cellular cathepsin H. This enzyme was acquired by phagosomes significantly faster than the other hydrolases tested. The second distinct site of lysosomal enzyme concentration was the late endosomes which contain the bulk of cathepsin S. The third and fourth large pools of hydrolases were found in two functionally distinct types of dense lysosomes, only one of which was found to be secreted in the presence of chloroquine or bafilomycin. Among this secreted pool was soluble furin, generally considered only as a membrane-bound trans-Golgi network resident protein. Thus, the organelles usually referred to as "lysosomes" in fact encompass a growing family of highly dynamic but functionally distinct endocytic organelles.     

Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.     

IkappaB is a substrate for a selective pathway of lysosomal proteolysis

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor kappa B (IkappaB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IkappaB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IkappaB is directly transported into isolated lysosomes in a process that requires binding of IkappaB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IkappaB uptake by lysosomes. Ubiquitination and phosphorylation of IkappaB are not required for its targeting to lysosomes. The lysosomal degradation of IkappaB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IkappaB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IkappaB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IkappaB is increased. There are both short- and long-lived pools of IkappaB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IkappaB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IkappaB degradation in lysosomes. This selective pathway of lysosomal degradation of IkappaB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor kappa B. In addition, the response of nuclear factor kappa B to several stimuli increases when this lysosomal pathway of proteolysis is activated.     

Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase

Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase.     

Proteasome inhibitors induce the association of Alzheimer's amyloid precursor protein with Hsc73

Amyloid precursor protein (APP) is a secretory membrane-bound protein that undergoes restrictive proteolysis and degradation with a short life span in the constitutive secretory pathway or in the endosomal/lysosomal compartment. The degradation machinery, including cellular trafficking and the restrictive cleavage of APP, is poorly understood. To gain further insight into the intracellular degradation mechanism of APP, we searched for effector proteins that interact with APP. We found that a cytosolic molecular chaperon, Hsc73, effectively interacts with the cytoplasmic domain of APP in the presence of proteasome inhibitors. Hsc73 binds to the cytoplasmic domain near the post-transmembrane region of APP and not to the KFERQ-related sequence, KFFEQ, at the C-terminal tail that is assumed to be the selective targeting signal for lysosomal proteolysis. The amounts of Hsc73 that bind to several APP species such as those found in pathological Familial Alzheimer's disease (FAD), Swedish, or Dutch type mutation, are almost identical, suggesting that an abnormal conformation around the secretory cleavage site or a pathological imbalance in APP processing are not irrelevant to the efficiency of Hsc73 binding.     

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.