scholarly journals Accumulation of rab4GTP in the Cytoplasm and Association with the Peptidyl-Prolyl Isomerase Pin1 during Mitosis

2000 ◽  
Vol 11 (7) ◽  
pp. 2201-2211 ◽  
Author(s):  
Lisya Gerez ◽  
Karin Mohrmann ◽  
Marcel van Raak ◽  
Mandy Jongeneelen ◽  
Xiao Zhen Zhou ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Subcellular Fractionation ◽  
Effector Proteins ◽  
Endocytic Pathway ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Gtpase Cycle ◽  
Mitotic Cells

Transport through the endocytic pathway is inhibited during mitosis. The mechanism responsible for this inhibition is not understood. Rab4 might be one of the proteins involved as it regulates transport through early endosomes, is phosphorylated by p34cdc2 kinase, and is translocated from early endosomes to the cytoplasm during mitosis. We investigated the perturbation of the rab4 GTPase cycle during mitosis. Newly synthesized rab4 was less efficiently targeted to membranes during mitosis. By subcellular fractionation of mitotic cells, we found a large increase of cytosolic rab4 in the active GTP-form, an increase not associated with the cytosolic rabGDP chaperone GDI. Instead, phosphorylated rab4 is in a complex with the peptidyl-prolyl isomerase Pin1 during mitosis, but not during interphase. Our results show that less efficient recruitment of rab4 to membranes and a bypass of the normal GDI-mediated retrieval of rab4GDP from early endosomes reduce the amount of rab4GTP on membranes during mitosis. We propose that phosphorylation of rab4 inhibits both the recruitment of rab4 effector proteins to early endosomes and the docking of rab4-containing transport vesicles. This mechanism might contribute to the inhibition of endocytic membrane transport during mitosis.

scholarly journals Rab10 Regulates Membrane Transport through Early Endosomes of Polarized Madin-Darby Canine Kidney Cells

2006 ◽  
Vol 17 (7) ◽  
pp. 3156-3175 ◽  
Author(s):  
Clifford M. Babbey ◽  
Nahid Ahktar ◽  
Exing Wang ◽  
Carlos Chih-Hsiung Chen ◽  
Barth D. Grant ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Mammalian Cells ◽  
Mdck Cells ◽  
Endocytic Pathway ◽  
Rab Proteins ◽  
Madin Darby Canine Kidney ◽  
Early Endosomes ◽  
Mutant Forms ◽  
Darby Canine Kidney ◽  
Canine Kidney

Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.

Degradation of phagosomal components in late endocytic organelles

1998 ◽  
Vol 111 (1) ◽  
pp. 141-148
Author(s):  
T.E. Tjelle ◽  
B. Saigal ◽  
M. Froystad ◽  
T. Berg
Keyword(s):  
Subcellular Fractionation ◽  
Endocytic Pathway ◽  
Phagocytic Cells ◽  
Fusion Event ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Efficient Communication ◽  
J774 Cells ◽  
Shift Technique

Phagosomes are formed when phagocytic cells ingest particles such as bacteria, viruses or synthetic beads of different kinds. The environment within the phagosome gradually changes to generate degradative conditions. These changes require multiple interactions between the maturing phagosomes and the endocytic and the biosynthetic pathway. The phagosomes probably communicate with endocytic organelles by a transient fusion event, often referred to as the ‘kiss-and-run’ hypothesis. We have studied the role of endocytic organelles in the phagocytic pathway of J774 cells, a mouse macrophage cell line. We have used magnetic Dynabeads coated with 125ITC-IgG and 125ITC-OVA as phagocytic probes and were able to isolate the phagosomal fraction by means of a magnet. To separate lysosomes from other organelles in the endocytic pathway we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three endocytic fractions corresponding to early endosomes (the light Percoll fraction), late endosomes (the dense Percoll fraction) and lysosomes (the gold fraction). We observed that the proteins linked to the ingested beads are initially cleaved in the phagosomes. This cleavage is inhibited by leupeptin, a thiol-protease inhibitor, and requires an acidic environment. However, efficient communication between the phagosomes and the endocytic pathway leads to the transfer of dissociated phagocytosed peptides of different sizes to late endosomes and lysosomes for further processing. Consequently, the late endosomes and the lysosomes may be involved in the degradation of phagocytosed compounds.

Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation

1996 ◽  
Vol 109 (12) ◽  
pp. 2905-2914 ◽  
Author(s):  
T.E. Tjelle ◽  
A. Brech ◽  
L.K. Juvet ◽  
G. Griffiths ◽  
T. Berg
Keyword(s):  
Lysosomal Enzymes ◽  
Degradation Products ◽  
Mannose Receptor ◽  
Subcellular Fractionation ◽  
Endocytic Pathway ◽  
Acid Hydrolases ◽  
Main Site ◽  
Isolation And Characterization ◽  
Late Endosomes ◽  
Early Endosomes

Although endosomal proteolysis has been reported (e.g. for peptide hormones and lysosomal enzymes), lysosomes are believed to be the main site of degradation in the endocytic pathway. We have studied the separate roles of lysosomes and prelysosomal endocytic organelles in the degradation of ovalbumin in J774 cells. The ovalbumin was labelled with 125I-tyramine cellobiose (125I-TC-ova). The labelled degradation products formed from this probe are trapped at the site of formation. To separate lysosomes efficiently from prelysosomal endocytic organelles we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three fractions that were sequentially involved in the endocytic pathway: a light Percoll fraction, a dense Percoll fraction and a gold fraction. The light Percoll fraction contained early endosomes since it was transferrin positive and received endocytic markers such as ovalbumin and horseradish peroxidase (HRP) early (< 5 minutes) after internalization. The dense Percoll fraction was transferrin negative, rab7 positive and received endocytic markers after 10–15 minutes of internalization. The gold-filled fraction was negative for both transferrin and rab7 but highly enriched in the lysosomal enzyme beta-hexosaminidase and was therefore defined as a lysosome. To study the role of endosomes and lysosomes in the degradation of endocytosed material we allowed the cells to take up (via the mannose receptor) 125I-TC-ova. It was found that the main degradation of 125I-TC-ova (measured as acid soluble radioactivity trapped in the organelle) took place in the late endosomes (and not in the lysosomes containing the bulk of the lysosomal enzymes). Our data therefore suggest that the late endosomes operate as an early lysosomal compartment. The terminal lysosomes may serve as storage bodies for acid hydrolases that may be called upon when needed (for instance during phagocytosis).

scholarly journals SNARE Function Is Not Involved in Early Endosome Docking

2008 ◽  
Vol 19 (12) ◽  
pp. 5327-5337 ◽  
Author(s):  
Ulf Geumann ◽  
Sina Victoria Barysch ◽  
Peer Hoopmann ◽  
Reinhard Jahn ◽  
Silvio O. Rizzoli
Keyword(s):  
Phosphatidyl Inositol ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Vitro Assay ◽  
Receptor Proteins ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Rab Effector

Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific Fab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.

scholarly journals A syntaxin 10–SNARE complex distinguishes two distinct transport routes from endosomes to the trans-Golgi in human cells

2008 ◽  
Vol 180 (1) ◽  
pp. 159-172 ◽  
Author(s):  
Ian G. Ganley ◽  
Eric Espinosa ◽  
Suzanne R. Pfeffer
Keyword(s):  
Human Cells ◽  
Endocytic Pathway ◽  
Snare Complex ◽  
Rab Gtpases ◽  
Protein Receptor ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Target Membrane ◽  
Guanosine Triphosphatase ◽  
Dependent Transport

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the Golgi after delivering lysosomal enzymes to the endocytic pathway. This process requires Rab9 guanosine triphosphatase (GTPase) and the putative tether GCC185. We show in human cells that a soluble NSF attachment protein receptor (SNARE) complex comprised of syntaxin 10 (STX10), STX16, Vti1a, and VAMP3 is required for this MPR transport but not for the STX6-dependent transport of TGN46 or cholera toxin from early endosomes to the Golgi. Depletion of STX10 leads to MPR missorting and hypersecretion of hexosaminidase. Mouse and rat cells lack STX10 and, thus, must use a different target membrane SNARE for this process. GCC185 binds directly to STX16 and is competed by Rab6. These data support a model in which the GCC185 tether helps Rab9-bearing transport vesicles deliver their cargo to the trans-Golgi and suggest that Rab GTPases can regulate SNARE–tether interactions. Importantly, our data provide a clear molecular distinction between the transport of MPRs and TGN46 to the trans-Golgi.

The peptidyl-prolyl isomerase SlrA contributes to virulence and adherence of Streptococcus pneumoniae

Pneumologie ◽  
2006 ◽  
Vol 60 (02) ◽  
Author(s):  
S Hammerschmidt ◽  
PV Adrian ◽  
C Albert ◽  
S Estevão ◽  
T Hoogenboezem ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase

scholarly journals Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin

1991 ◽  
Vol 266 (4) ◽  
pp. 2474-2479
Author(s):  
T F Holzman ◽  
D A Egan ◽  
R Edalji ◽  
R L Simmer ◽  
R Helfrich ◽  
...  
Keyword(s):  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Preliminary Characterization

scholarly journals Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

1993 ◽  
Vol 177 (3) ◽  
pp. 583-596 ◽  
Author(s):  
P Romagnoli ◽  
C Layet ◽  
J Yewdell ◽  
O Bakke ◽  
R N Germain
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

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