The ARF guanine nucleotide exchange factor GBF1 is targeted to Golgi membranes through a PIP-binding domain

Abstract

ADP-ribosylation factors (ARF) GTPases are activated by guanine nucleotide exchange factors (GEFs) to support cellular homeostasis. Key to understanding spatio-temporal regulation of ARF signaling is the mechanism of GEF recruitment to membranes. Small GEFs are recruited through phosphoinositide (PIP) binding by a pleckstrin homology (PH) domain downstream from the catalytic Sec7 domain (Sec7d). The large GEFs lack PH domains, and their recruitment mechanisms are poorly understood. We probed Golgi recruitment of GBF1, a GEF catalyzing ARF activation required for Golgi homeostasis. We show that the homology downstream of Sec7d-1 (HDS1) regulates Golgi recruitment of GBF1. We document that GBF1 binds phosphoinositides, preferentially PI3P, PI4P and PI(4,5)P, and that lipid binding requires the HDS1 domain. Mutations within HDS1 that reduce GBF1 binding to specific PIPs inhibit GBF1 targeting to Golgi membranes in cells. Our data imply that HDS1 and PH domains are functionally analogous in that each uses lipid-based membrane information to regulate GEF recruitment. Lipid-based recruitment of GBF1 extends the paradigm of lipid regulation to small and large GEFs and suggests that lipid-based mechanisms evolved early during GEF diversification. This article has an associated First Person interview with the first author of the paper.

A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1

“…It is known that replication organelles of picornaviruses, including those of poliovirus, are highly enriched in PI4P (13). Yet, the mutation LF926AA in the HDS1 domain of GBF1, previously found to impair targeting of GBF1 to PIP-containing membranes (35), did not decrease the replication of the 3A-2 mutant and even somewhat outperformed the positive control. The most severe negative effect on the replication of the 3A-2 replicon was observed with the L1246R mutation of a conserved amino acid in the HDS2 domain of GBF1.…”

“…Previously, we generated a series of mutations in the highly conserved regions of the HDS1, HDS2, and HDS3 domains of GBF1 targeting the most conserved residues. The HDS1 domain was recently found to target GBF1 to phosphoinositide phosphate (PIP)-enriched membranes, and the LF926AA mutation (number corresponds to the position of the first amino acid changed in the GBF1 sequence) disrupted this property and inhibited GBF1 function in secretion (35). Similarly, the HDS2 mutants L1246R, LF1266AA, and RDR1168AAA were severely compromised in supporting the cellular secretory pathway and targeting to the Golgi, while the HDS2 mutant LMK1135AAA behaved like a positive control in a secretion assay (23).…”

“…This suggests that there may be a significant excess of GBF1 available in cells, at least in the conventional cell culture models used for the study of picornaviruses, or that there is an axillary mechanism targeting GBF1 to the replication membranes that may compensate for the weakened 3A-GBF1 interaction. We previously found that mutations in the conserved regions of GBF1 located in the C-terminal noncatalytic domains of the protein prevent its targeting to Golgi membranes and inhibit GBF1 function in the secretory pathway (35,53). When we tested such mutants in poliovirus replication, all fully supported replication of the wild-type replicon, but several C-terminal GBF1 mutants were severely compromised in supporting the replication of a 3A-2 replicon carrying a mutation that strongly diminishes the 3A-GBF1 interaction (6,7,42).…”

The Guanine Nucleotide Exchange Factor GBF1 Participates in Rotavirus Replication

“…Distinct domains of GBF1 are essential for rotavirus replication. To explore the potential involvement of the different GBF1 domains in rotavirus progeny production, we compared the replication of the virus in HEK293 cells transfected with the GBF1 construct containing the A795E mutation (GBF1/795) that confers resistance to BFA (47,58) or with a series of mutant GBF1/795 proteins ( Fig. 12A) (59).…”

“…In addition, GBF1 contains five noncatalytic domains: the N-terminal dimerization and cyclophilin binding (DCB) domain, the homology domain upstream of Sec7 (HUS), and three C-terminal homology domains downstream of Sec7 (HDS1 to -3) (33,36). The functions of these noncatalytic domains are not well understood, but the N-terminal DCB and HUS domains have been implicated in inter-and intramolecular interactions important for GBF1 dimerization and its association with membranes (43,44), while the HDS1 to -3 domains have been suggested to facilitate GBF1 association with membranes (45)(46)(47)(48).…”