scholarly journals Soi3p/Rav1p Functions at the Early Endosome to Regulate Endocytic Trafficking to the Vacuole and Localization of Trans-Golgi Network Transmembrane Proteins

2004 ◽  
Vol 15 (7) ◽  
pp. 3196-3209 ◽  
Author(s):  
György Sipos ◽  
Jason H. Brickner ◽  
E.J. Brace ◽  
Linyi Chen ◽  
Alain Rambourg ◽  
...  
Keyword(s):  
Vacuolar Atpase ◽  
Early Endosome ◽  
Trans Golgi Network ◽  
Peripheral Protein ◽  
Early Endosomes ◽  
Localization Signal ◽  
Prevacuolar Compartment ◽  
Gradient Fractionation ◽  
Vacuolar Protein ◽  
Golgi Network

SOI3 was identified by a mutation, soi3-1, that suppressed a mutant trans-Golgi network (TGN) localization signal in the Kex2p cytosolic tail. SOI3, identical to RAV1, encodes a protein important for regulated assembly of vacuolar ATPase. Here, we show that Soi3/Rav1p is required for transport between the early endosome and the late endosome/prevacuolar compartment (PVC). By electron microscopy, soi3-1 mutants massively accumulated structures that resembled early endosomes. soi3Δ mutants exhibited a kinetic delay in transfer of the endocytic tracer dye FM4-64, from the 14°C endocytic intermediate to the vacuole. The soi3Δ mutation delayed vacuolar degradation but not internalization of the a-factor receptor Ste3p. By density gradient fractionation, Soi3/Rav1p associated as a peripheral protein with membranes of a density characteristic of early endosomes. The soi3 null mutation markedly reduced the rate of Kex2p transport from the TGN to the PVC but had no effect on vacuolar protein sorting or cycling of Vps10p. These results suggest that assembly of vacuolar ATPase at the early endosome is required for transport of both Ste3p and Kex2p from the early endosome to the PVC and support a model in which cycling through the early endosome is part of the normal itinerary of Kex2p and other TGN-resident proteins.

scholarly journals Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

2008 ◽  
Vol 19 (11) ◽  
pp. 4826-4836 ◽  
Author(s):  
Mohamed E. Abazeed ◽  
Robert S. Fuller
Keyword(s):  
Binding Proteins ◽  
Adaptor Protein ◽  
Early Endosome ◽  
Dominant Negative ◽  
Factor Binding ◽  
Trans Golgi Network ◽  
Direct Pathway ◽  
Prevacuolar Compartment ◽  
Golgi Network ◽  
Adp Ribosylation Factor

Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome.

scholarly journals Allele-specific suppression of a defective trans-Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins.

1996 ◽  
Vol 16 (11) ◽  
pp. 6208-6217 ◽  
Author(s):  
K Redding ◽  
J H Brickner ◽  
L G Marschall ◽  
J W Nichols ◽  
R S Fuller
Keyword(s):  
Point Mutations ◽  
Mutant Form ◽  
Trans Golgi Network ◽  
Gal1 Promoter ◽  
Allele Specific ◽  
Localization Signal ◽  
Dipeptidyl Aminopeptidase ◽  
Vacuolar Protein ◽  
Golgi Network ◽  
Recessive Defect

Kex2 protease (Kex2p) and Ste13 dipeptidyl aminopeptidase (Ste13p) are required in Saccharomyces cerevisiae for maturation of the alpha-mating factor in a late Golgi compartment, most likely the yeast trans-Golgi network (TGN). Previous studies identified a TGN localization signal (TLS) in the C-terminal cytosolic tail of Kex2p consisting of Tyr-713 and contextual sequences. Further analysis of the Kex2p TLS revealed similarity to the Ste13p TLS. Mutation of the Kex2p TLS results in transport of Kex2p to the vacuole by default. When expression of a GAL1 promoter-driven KEX2 gene is shut off in MAT(alpha) cells, the TGN becomes depleted of Kex2p, resulting in a gradual decline in mating competence which is greatly accelerated by TLS mutations. To identify the genes involved in localization of Kex2p, we isolated second-site suppressors of the rapid loss of mating competence observed upon shutting off expression of a TLS mutant form of Kex2p (Y713A). Seven of 58 suppressors were allele specific, suppressing point mutations at Tyr-713 but not deletions of the TLS or entire C-terminal cytosolic tail. By linkage analysis, the allele-specific suppressors defined three genetic loci, SOI1, S0I2, and S0I3. Pulse-chase analysis demonstrated that these suppressors increased net TGN retention of both Y713A Kex2p and a Ste13p-Pho8p fusion protein containing a point mutation in the Ste13p TLS. SOI1 suppressor alleles reduced the efficiency of localization of wild-type Kex2p to the TGN, implying an impaired ability to discriminate between the normal TLS and a mutant TLS. soi1 mutants also exhibited a recessive defect in vacuolar protein sorting. Suppressor alleles of S0I2 were dominant. These results suggest that the SOI1 and S0I2 genes encode regulators or components of the TLS recognition machinery.

scholarly journals P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

2008 ◽  
Vol 19 (8) ◽  
pp. 3526-3535 ◽  
Author(s):  
Ke Liu ◽  
Kavitha Surendhran ◽  
Steven F. Nothwehr ◽  
Todd R. Graham
Keyword(s):  
Protein Transport ◽  
Early Endosome ◽  
Coated Vesicle ◽  
Transport Pathways ◽  
Vesicle Budding ◽  
Trans Golgi Network ◽  
Conditional Allele ◽  
Early Endosomes ◽  
Clathrin Adaptor ◽  
Golgi Network

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Δ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.

scholarly journals The role of the AP-1 adaptor complex in trafficking between the trans-Golgi Network and endosomal system

2005 ◽  
Author(s):  
◽  
Christopher Foote
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Early Endosome ◽  
Trans Golgi Network ◽  
Retrieval Mechanism ◽  
Prevacuolar Compartment ◽  
Golgi Network ◽  
In Transit ◽  
Endosomal System ◽  
Kinetics Of

In Saccharomyces cerevisiae it is generally accepted that there are two routes for trafficking of proteins from the trans-Golgi network (TGN) to the vacuole. One involves direct transport from the TGN to the vacuole. The second involves transport from the TGN to the prevacuolar compartment (PVC) via GGA coated vesicles, followed by PVC to vacuole transport. We propose that there is a third route. This route entails transit from the TGN to the early endosome (EE), followed by delivery to the PVC and vacuole. In support of an alternative route, the processing kinetics of A(F[arrow]A)-ALP are not affected by mutations in the GGA proteins. This is in contrast to proteins that use the GGA pathway, as their delivery to the vacuole is significantly slowed when GGA function is ablated. Further support of an EE itinerary is the observation that A(F[arrow]A)-ALP co localizes with the lipophilic dye, FM4-64 at a time when the dye is associated with the EE. Disruption of the AP-1 vesicle coat complex leads to an accelerated processing of A(F[arrow]A)-ALP. Appending the region of A(F[arrow]A)-ALP that interacts with AP-1 to Cps1p delays its progress to the vacuole. These results are consistent with a model in which A(F[arrow]A)-ALP passes through the EE in transit to the vacuole. A(F[arrow]A)-ALP physically interacts with AP-1, and this interaction delays its delivery to the vacuole. Data presented in this thesis suggests that in Saccharomyces cerevisiae AP-1 functions as a retrieval mechanism from the EE to the TGN.

scholarly journals Characterization of RIN3 as a Guanine Nucleotide Exchange Factor for the Rab5 Subfamily GTPase Rab31

2011 ◽  
Vol 286 (27) ◽  
pp. 24364-24373 ◽  
Author(s):  
Hiroaki Kajiho ◽  
Kyoko Sakurai ◽  
Tomohiro Minoda ◽  
Manabu Yoshikawa ◽  
Satoshi Nakagawa ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Endocytic Pathway ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Trans Golgi Network ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Early Endosomes ◽  
Vacuolar Protein ◽  
Golgi Network

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.

scholarly journals SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1–positive Early Endosome/Trans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2592-2603 ◽  
Author(s):  
Waka Natsume ◽  
Kenji Tanabe ◽  
Shunsuke Kon ◽  
Naomi Yoshida ◽  
Toshio Watanabe ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Adaptor Proteins ◽  
Early Endosome ◽  
Dependent Manner ◽  
Transferrin Receptors ◽  
Gtpase Activating Protein ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Golgi Network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

Multimeric connexin interactions prior to the trans-Golgi network

2001 ◽  
Vol 114 (22) ◽  
pp. 4013-4024
Author(s):  
Jayasri Das Sarma ◽  
Rita A. Meyer ◽  
Fushan Wang ◽  
Valsamma Abraham ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Brefeldin A ◽  
Dominant Negative ◽  
Alveolar Epithelial Cells ◽  
Trans Golgi Network ◽  
Alveolar Epithelial ◽  
C Terminus ◽  
Gradient Fractionation ◽  
Golgi Network ◽  
Nih 3T3 ◽  
Gap Junction Hemichannels

Cells that express multiple connexins have the capacity to form heteromeric (mixed) gap junction hemichannels. We used a dominant negative connexin construct, consisting of bacterial β-galactosidase fused to the C terminus of connexin43 (Cx43/β-gal), to examine connexin compatibility in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool induced by Cx43/β-gal colocalized with a medial Golgi apparatus marker and was readily disassembled by treatment with brefeldin A. This was unexpected, since previous studies indicated that Cx43 assembly into hexameric hemichannels occurs in the trans-Golgi network (TGN) and is sensitive to brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a subhexameric complex. Cx43/β-gal also specifically interacted with Cx46, but not Cx32, consistent with the ability of Cx43/β-gal to simultaneously inhibit multiple connexins. We confirmed that interactions between Cx43/β-gal and Cx46 reflect the ability of Cx43 and Cx46 to form heteromeric complexes, using HeLa and alveolar epithelial cells, which express both connexins. In contrast, ROS osteoblastic cells, which differentially sort Cx43 and Cx46, did not form Cx43/Cx46 heteromers. Thus, cells have the capacity to regulate whether or not compatible connexins intermix.

scholarly journals Transition of Galactosyltransferase 1 from Trans-Golgi Cisterna to the Trans-Golgi Network Is Signal Mediated

2006 ◽  
Vol 17 (12) ◽  
pp. 5153-5162 ◽  
Author(s):  
Beat E. Schaub ◽  
Bea Berger ◽  
Eric G. Berger ◽  
Jack Rohrer
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Tail ◽  
Time Lapse ◽  
Trans Golgi Network ◽  
Golgi Cisterna ◽  
Confocal Fluorescence ◽  
Rapid Accumulation ◽  
Gradient Fractionation ◽  
Golgi Network ◽  
Complex Glycan

The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate β1,4-galactosyltransferase 1 (galT) and α2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT–siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.

Sign in / Sign up

Export Citation Format

Share Document