Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell.
Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.
Zhu
Huaiping
H
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Zhu
Guangyu
G
Liu
Jay
J
Liang
Zhimin
Z
Zhang
Xuejun C
XC
Li
Guangpu
G
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D008561:Membrane Fusion; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D050505:Mutant Proteins; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins
J Cell Biol. 2000 Apr 3;149(1):95-110
10747090
Nat Struct Mol Biol. 2007 May;14(5):406-12
17450153
Biochem J. 2001 May 1;355(Pt 3):681-9
11311130
J Mol Biol. 2001 Jun 29;310(1):141-56
11419942
Mol Biol Cell. 2001 Jul;12(7):2219-28
11452015
Dev Cell. 2001 Jul;1(1):73-82
11703925
J Biol Chem. 2002 Feb 1;277(5):3412-8
11733506
Nat Immunol. 2004 Aug;5(8):844-52
15235600
Cell. 2004 Sep 3;118(5):607-17
15339665
Nat Struct Mol Biol. 2004 Oct;11(10):975-83
15378032
EMBO J. 2004 Oct 13;23(20):3909-17
15457209
Cell. 1991 Mar 8;64(5):915-25
1900457
Cell. 1992 Sep 4;70(5):715-28
1516130
J Biol Chem. 1993 Nov 15;268(32):24475-80
8226999
EMBO J. 1994 Mar 15;13(6):1287-96
8137813
J Biol Chem. 1994 May 20;269(20):14631-5
8182071
J Biol Chem. 1995 Mar 10;270(10):5048-56
7890612
Cell. 1997 Sep 19;90(6):1149-59
9323142
Trends Cell Biol. 2006 Jan;16(1):27-35
16330212
J Biol Chem. 2006 Mar 10;281(10):6874-83
16407276
Nat Struct Mol Biol. 2006 Mar;13(3):264-71
16462746
Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2659-64
16533754
Cell. 2006 Mar 24;124(6):1183-95
16499958
Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):11821-7
16882731
Nat Rev Mol Cell Biol. 2001 Feb;2(2):107-17
11252952