scholarly journals Redundant Roles of BIG2 and BIG1, Guanine-Nucleotide Exchange Factors for ADP-Ribosylation Factors in Membrane Traffic between the trans-Golgi Network and Endosomes

2008 ◽  
Vol 19 (6) ◽  
pp. 2650-2660 ◽  
Author(s):  
Ray Ishizaki ◽  
Hye-Won Shin ◽  
Hiroko Mitsuhashi ◽  
Kazuhisa Nakayama
Keyword(s):  
Membrane Traffic ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Recycling Endosomes ◽  
Adp Ribosylation ◽  
Trans Golgi Network ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Golgi Network

BIG2 and BIG1 are closely related guanine-nucleotide exchange factors (GEFs) for ADP-ribosylation factors (ARFs) and are involved in the regulation of membrane traffic through activating ARFs and recruiting coat protein complexes, such as the COPI complex and the AP-1 clathrin adaptor complex. Although both ARF-GEFs are associated mainly with the trans-Golgi network (TGN) and BIG2 is also associated with recycling endosomes, it is unclear whether BIG2 and BIG1 share some roles in membrane traffic. We here show that knockdown of both BIG2 and BIG1 by RNAi causes mislocalization of a subset of proteins associated with the TGN and recycling endosomes and blocks retrograde transport of furin from late endosomes to the TGN. Similar mislocalization and protein transport block, including furin, were observed in cells depleted of AP-1. Taken together with previous reports, these observations indicate that BIG2 and BIG1 play redundant roles in trafficking between the TGN and endosomes that involves the AP-1 complex.

scholarly journals Arf3 Is Activated Uniquely at the trans-Golgi Network by Brefeldin A-inhibited Guanine Nucleotide Exchange Factors

2010 ◽  
Vol 21 (11) ◽  
pp. 1836-1849 ◽  
Author(s):  
Florin Manolea ◽  
Justin Chun ◽  
David W. Chen ◽  
Ian Clarke ◽  
Nathan Summerfeldt ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Temperature Sensitive ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Membrane Composition ◽  
Trans Golgi Network ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Golgi Membranes ◽  
Golgi Network

It is widely assumed that class I and II Arfs function interchangeably throughout the Golgi complex. However, we report here that in vivo, Arf3 displays several unexpected properties. Unlike other Golgi-localized Arfs, Arf3 associates selectively with membranes of the trans-Golgi network (TGN) in a manner that is both temperature-sensitive and uniquely dependent on guanine nucleotide exchange factors of the BIGs family. For example, BIGs knockdown redistributed Arf3 but not Arf1 from Golgi membranes. Furthermore, shifting temperature to 20°C, a temperature known to block cargo in the TGN, selectively redistributed Arf3 from Golgi membranes. Arf3 redistribution occurred slowly, suggesting it resulted from a change in membrane composition. Arf3 knockdown and overexpression experiments suggest that redistribution is not responsible for the 20°C block. To investigate in more detail the mechanism for Arf3 recruitment and temperature-dependent release, we characterized several mutant forms of Arf3. This analysis demonstrated that those properties are readily separated and depend on pairs of residues present at opposite ends of the protein. Furthermore, phylogenetic analysis established that all four critical residues were absolutely conserved and unique to Arf3. These results suggest that Arf3 plays a unique function at the TGN that likely involves recruitment by a specific receptor.

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.

Arf, Sec7 and Brefeldin A: a model towards the therapeutic inhibition of guanine nucleotide-exchange factors

2005 ◽  
Vol 33 (6) ◽  
pp. 1265-1268 ◽  
Author(s):  
M. Zeghouf ◽  
B. Guibert ◽  
J.-C. Zeeh ◽  
J. Cherfils
Keyword(s):  
Exchange Reaction ◽  
G Proteins ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Small G Proteins

GEFs (guanine nucleotide-exchange factors), which stimulate GDP dissociation from small G-proteins, are pivotal regulators of signalling pathways activated by small G-proteins. In the case of Arf proteins, which are major regulators of membrane traffic in the cell and have recently been found to be involved in an increasing number of human diseases, GDP/GTP exchange is stimulated by GEFs that carry a catalytic Sec7 domain. Recent structural results captured snapshots of the exchange reaction, revealing that Sec7 domains secure Arf-GDP to membranes before nucleotide exchange takes place, taking advantage of a built-in structural device in Arf proteins that couples their affinity for membranes to the nature of the bound nucleotide. One of the Arf–Sec7 intermediates was trapped by BFA (Brefeldin A), an uncompetitive inhibitor of Arf activation that has been instrumental in deciphering the molecular principles of membrane traffic at the Golgi. BFA targets a low-affinity Arf–Sec7 intermediate of the exchange reaction. It binds at the Arf-GDP/Sec7 interface, thus freezing the complex in an abortive conformation that cannot proceed to nucleotide dissociation. In the cell, this results in the specific inhibition of Arf1 by a subset of its GEFs, and the efficient and reversible block of membrane traffic at the Golgi. The mechanism of BFA leads to the concept of ‘interfacial inhibition’, in which a protein–protein interaction of therapeutic interest is stabilized, rather than impaired, by a drug. Up-regulated activity of small G-proteins is involved in various human diseases, making their GEFs attractive candidates to interrupt specifically the corresponding signalling pathway. Interfacial inhibitors are proposed as an alternative to competitive inhibitors that may be explored for their inhibition.

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