The amphipathic helices of Arfrp1 and Arl14 are sufficient to determine subcellular localizations

Abstract

The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors (GEFs). By promoting GTP binding, GEFs induce conformational changes of Arf proteins exposing their N-terminal amphipathic helices, which then insert into the membranes to stabilize the membrane association process. Here, we found the N-terminal amphipathic motifs of the Golgi-localized Arf family protein, Arfrp1, and the endosome- and plasma membrane-localized Arf family protein, Arl14, play critical roles in spatial determination. Exchanging the amphipathic helix motifs between these two Arf proteins causes the switch of their localizations. Moreover, the amphipathic helices of Arfrp1 and Arl14 are sufficient for cytosolic proteins to be localized into a specific cellular compartment. The spatial determination mediated by the Arfrp1 helix requires its binding partner Sys1. In addition, the residues that are required for the acetylation of the Arfrp1 helix and the myristoylation of the Arl14 helix are important for the specific subcellular localization. Interestingly, Arfrp1 and Arl14 are recruited to their specific cellular compartments independent of GTP binding. Our results demonstrate that the amphipathic motifs of Arfrp1 and Arl14 are sufficient for determining specific subcellular localizations in a GTP-independent manner, suggesting the membrane association and activation of some Arf proteins are uncoupled.

ARL14 as a Prognostic Biomarker in Non-Small Cell Lung Cancer

“…13 The ARL14 protein is involved in apoptosis signaling and several regulatory pathways. [25][26][27][28][29][30] Its homologous similar genes play an important role in human tumors; for example, ARF4 is involved in breast cancer cell migration as reported by Jang et al 31 Our study indicated that ARL14 protein appeared to be a promising candidate as a therapeutic target and prognostic predictor. Using bioinformatics analysis based on online datasets, survival analysis revealed the prognostic value of ARL14 in NSCLC.…”

“… 13 The ARL14 protein is involved in apoptosis signaling and several regulatory pathways. 25–30 Its homologous similar genes play an important role in human tumors; for example, ARF4 is involved in breast cancer cell migration as reported by Jang et al 31 …”

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

“…To test whether PRRC1 can be recruited to ERESs in a GTP-dependent manner, we performed a digitonin-permeabilized cell assay ( 35 ). HeLa cells were permeabilized, salt washed, and incubated with cytosol prepared from HEK293T cells expressing PRRC1-HA in the presence of GTP or GMPPNP.…”

“…Quantifications of the total fluorescence of Sec31A and Gogin97 were performed as described using ImageJ ( 46 ). Permeabilized cell assays were performed as described previously ( 35 ).…”

Binding with heat shock cognate protein HSC70 fine-tunes the Golgi association of the small GTPase ARL5B

“…S2C). The deletion of the amphipathic helix J o u r n a l P r e -p r o o f of ARLs like ARFRP1and ARL14 or mutations in the myristoylation site of ARL5B and ARL1 was previously shown to have a similar localization (18,48,49).…”