scholarly journals Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors.

1995 ◽  
Vol 129 (1) ◽  
pp. 81-97 ◽  
Author(s):  
F Garcia-del Portillo ◽  
B B Finlay
Keyword(s):  
Epithelial Cells ◽  
Salmonella Typhimurium ◽  
Intracellular Trafficking ◽  
Secretory Pathway ◽  
Bacterial Pathogen ◽  
Lysosomal Membrane ◽  
Membrane Glycoproteins ◽  
Mannose 6 Phosphate ◽  
Secondary Lysosomes ◽  
Lysosomal Membrane Glycoprotein

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.

scholarly journals Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles.

1988 ◽  
Vol 107 (6) ◽  
pp. 2491-2501 ◽  
Author(s):  
H J Geuze ◽  
W Stoorvogel ◽  
G J Strous ◽  
J W Slot ◽  
J E Bleekemolen ◽  
...  
Keyword(s):  
Lysosomal Membrane ◽  
Membrane Glycoprotein ◽  
Horse Radish Peroxidase ◽  
Western Blots ◽  
Lysosomal Membrane Proteins ◽  
Mannose 6 Phosphate ◽  
Lysosomal Membrane Glycoprotein ◽  
Endocytic Vesicles ◽  
Kinetics Of ◽  
Combined Data

The intracellular distributions of the cation-independent mannose 6-phosphate receptor (MPR) and a 120-kD lysosomal membrane glycoprotein (lgp120) were studied in rat hepatoma cells. Using quantitative immunogold cytochemistry we found 10% of the cell's MPR located at the cell surface. In contrast, lgp120 was not detectable at the plasma membrane. Intracellularly, MPR mainly occurred in the trans-Golgi reticulum (TGR) and endosomes. lgp120, on the other hand, was confined to endosomes and lysosomes. MPR was present in both endosomal tubules and vacuoles, whereas lgp120 was confined to the endosomal vacuoles. In cells incubated for 5-60 min with the endocytic tracer cationized ferritin, four categories of endocytic vacuoles could be discerned, i.e., vacuoles designated MPR+/lgp120-, MPR+/lgp120+, MPR-/lgp120+, and vacuoles nonimmunolabeled for MPR and lgp120. Tracer first reached MPR+/lgp120-, then MPR+/lgp120+, and finally MPR-/lgp120+ vacuoles, which are assumed to represent lysosomes. To study the kinetics of appearance of endocytic tracers in MPR-and/or lgp120-containing pools in greater detail, cells were allowed to endocytose horse-radish peroxidase (HRP) for 5-90 min. The reduction in detectability of MPR and lgp120 antigenicity on Western blots, due to treatment of cell homogenates with 3'3-diaminobenzidine, was followed in time. We found that HRP reached the entire accessible pool of MPR almost immediately after internalization of the tracer, while prolonged periods of time were required for HRP to maximally access lgp120. The combined data suggest that MPR+/lgp120+ vacuoles are endocytic vacuoles, intermediate between MPR+/lgp120-endosomes and MPR-/lgp120+ lysosomes, and represent the site where MPR is sorted from lgp120 destined for lysosomes. We propose that MPR is sorted from lgp120 by selective lateral distribution of the receptor into the tubules of this compartment, resulting in the retention of lgp120 in the vacuoles and the net transport of lgp120 to lysosomes.

The majority of clathrin coated vesicles from lactating rabbit mammary gland arises from the secretory pathway

1999 ◽  
Vol 112 (22) ◽  
pp. 4089-4100 ◽  
Author(s):  
A. Pauloin ◽  
S.A. Tooze ◽  
I. Michelutti ◽  
S. Delpal ◽  
M. Ollivier-Bousquet
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Bovine Brain ◽  
Mammary Epithelial ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Secretory Vesicles ◽  
Mannose 6 Phosphate

Clathrin coated vesicles were isolated from lactating rabbit mammary gland by differential centrifugation, centrifugation on (2)H2O-sucrose cushions and Sephacryl S-1000 chromatography. Mammary epithelial cells contain an unexpectedly high quantity of clathrin coated vesicles which appear heterogeneous in size, with a mean diameter of 95.9+/-10.5 nm and a density of 1.23 g × ml(−1). Analysis of clathrin coated vesicle adaptor composition by SDS-PAGE and western blot showed that only approximately 5–10% of total APs consist of AP-2 in isolated mammary gland clathrin coated vesicles whereas it represents approximately 70% of the total APs from bovine brain clathrin coated vesicles. Cargo molecules known to be transcytosed such as IgG, IgA, and the pIgR were detected in the clathrin coated vesicles, indicating that part of this vesicle population is involved in transcytotic pathways. However, as the vast majority of the clathrin coated vesicles contained AP-1, it was likely that these clathrin coated vesicles were involved in the secretory pathway. Relatively high quantities of furin and cation-independent mannose 6-phosphate receptor were detected in mammary clathrin coated vesicles. By immuno electron microscopy, AP-1 and the cation-independent mannose 6-phosphate receptor were localized in Golgi-associated vesicles and on the membrane of secretory vesicles. The presence of AP-1 in the coat patches on the membrane of secretory vesicles containing casein micelles, and the presence of alpha(s1)-casein in mammary gland clathrin coated vesicles, support a role for AP-1 in the maturation of secretory vesicles. Our data pinpoint the importance of clathrin coated vesicles in lactating mammary epithelial cells, and suggest these vesicles are involved in the transcytotic pathway, in sorting at the trans-Golgi network and in the biogenesis of casein-containing secretory vesicles.

scholarly journals An endogenous MDCK lysosomal membrane glycoprotein is targeted basolaterally before delivery to lysosomes.

1991 ◽  
Vol 115 (6) ◽  
pp. 1573-1584 ◽  
Author(s):  
I R Nabi ◽  
A Le Bivic ◽  
D Fambrough ◽  
E Rodriguez-Boulan
Keyword(s):  
Mdck Cells ◽  
Lysosomal Membrane ◽  
Endocytic Pathway ◽  
Membrane Glycoprotein ◽  
Membrane Glycoproteins ◽  
Degradation Rates ◽  
Early Endosomes ◽  
The Family ◽  
Basolateral Plasma Membrane ◽  
Lysosomal Membrane Glycoprotein

Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.

Sign in / Sign up

Export Citation Format

Share Document