scholarly journals The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach

1992 ◽  
Vol 103 (3) ◽  
pp. 785-796 ◽  
Author(s):  
C. Antony ◽  
C. Cibert ◽  
G. Geraud ◽  
A. Santa Maria ◽  
B. Maro ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
G Protein ◽  
Cell Lines ◽  
Binding Protein ◽  
Rab Proteins ◽  
Gtp Binding Protein ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Golgi Network ◽  
Exocytic Pathway

A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exocytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20 degrees C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.

scholarly journals A novel GTP-binding protein–adaptor protein complex responsible for export of Vangl2 from the trans Golgi network

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Yusong Guo ◽  
Giulia Zanetti ◽  
Randy Schekman
Keyword(s):  
Mammalian Cells ◽  
Planar Cell Polarity ◽  
Binding Protein ◽  
Adaptor Protein ◽  
Gtp Binding Protein ◽  
Epithelial Surface ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Signaling Receptors ◽  
Golgi Network

Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.

scholarly journals Role of the Rab GTP-Binding Protein Ypt3 in the Fission Yeast Exocytic Pathway and Its Connection to Calcineurin Function

2002 ◽  
Vol 13 (8) ◽  
pp. 2963-2976 ◽  
Author(s):  
Hong Cheng ◽  
Reiko Sugiura ◽  
Wenlian Wu ◽  
Masaaki Fujita ◽  
Yabin Lu ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Binding Protein ◽  
Leader Peptide ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Gtp Binding Protein ◽  
Cytoplasmic Staining ◽  
Cellular Processes ◽  
Gtp Binding ◽  
Exocytic Pathway

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed thatypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1+ leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.

scholarly journals The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.

1989 ◽  
Vol 109 (3) ◽  
pp. 1015-1022 ◽  
Author(s):  
R A Bacon ◽  
A Salminen ◽  
H Ruohola ◽  
P Novick ◽  
S Ferro-Novick
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Transport Defect

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.

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