The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors (GEFs). 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 acetylation of the Arfrp1 helix and the myristoylatio...[ Read more ]

The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors (GEFs). 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 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. In sum, our results demonstrate that the GTP binding and membrane association are of Arfrp1 and Arl14 are uncoupled. The amphipathic motifs of Arfrp1 and Arl14 are sufficient for them to be located to specific cellular compartments in a GTP-independent manner.

The insulin-like growth factor 2 (IGF2) plays critical roles in cell proliferation, growth, migration, differentiation and survival. Despite its importance, the molecular mechanisms mediating secretion of IGF2 remain unclear. Here we utilized a Retention Using Selective Hook (RUSH) system to analyze molecular mechanisms that regulate secretion of IGF2. We found that a type I transmembrane protein, TMED10, is essential for secretion of IGF2. Further analyses indicate that the residues from position 112 to position 140 in IGF2 is important for the secretion of IGF2 and these residues directly interact with the GOLD domain of TMED10. We then reconstituted release of IGF2 into COPII vesicles and this assay suggests that TMED10 regulates the packaging of IGF2 in COPII vesicles to be efficiently delivered to the Golgi. Moreover, we found that knockdown of TMED10 causes defects of secretion of IGF2 from mouse myoblast C2C12 cells and induces defects in C2C12 cell differentiation and myotube formation. Adding purified IGF2 into the differentiation medium rescue these defects in TMED10 knockdown C2C12 cells. Our analyses indicate that TMED10 functions as a cargo receptor to mediate packaging of IGF2 into COPII vesicles to regulate IGF2 secretion for myoblast differentiation.