Abstract
ARL5B, an ARF-like small GTPase localized to the trans-Golgi, is known for regulating endosome-Golgi trafficking and promoting the migration and invasion of breast cancer cells. Although a few interacting partners have been identified, the mechanism of the shuttling of ARL5B between the Golgi membrane and the cytosol is still obscure. Here, using GFP-binding protein (GBP) pull-down followed by mass spectrometry, we identified heat shock cognate protein (HSC70) as an additional interacting partner of ARL5B. Our pull-down and isothermal titration calorimetry (ITC)-based studies suggested that HSC70 binds to ARL5B in an ADP-dependent manner. Additionally, we showed that the N-terminal helix and the nucleotide status of ARL5B contribute to its recognition by HSC70. The confocal microscopy and cell fractionation studies in MDA-MB-231 breast cancer cells revealed that the depletion of HSC70 reduces the localization of ARL5B to the Golgi. Using invitro reconstitution approach, we provide evidence that HSC70 fine-tunes the association of ARL5B with Golgi membrane. Finally, we demonstrated that the interaction between ARL5B and HSC70 is important for the localization of cation independent mannose-6-phosphate receptor (CIMPR) at Golgi. Collectively, we propose a mechanism by which HSC70, a constitutively expressed chaperone, modulates the Golgi association of ARL5B, which in turn has implications for the Golgi-associated functions of this GTPase.
Highlights
ARF-like proteins (ARLs) belong to the ARF family of small GTPases and perform diverse functions such as membrane trafficking, tubulin assembly, lysosome positioning, etc. [1,2,3]
Using peptide mass fingerprinting (PMF), we identified the protein as Heat Shock Cognate Protein 70 (HSC70) (HSP7C) with an expect value
ARF-like 5B (ARL5B) is previously shown to regulate the transport of cargoes between the Golgi and endosomes by recruiting proteins such as Adaptor protein complex; AP4, Golgi-associated retrograde protein (GARP), and Lamtor1, a subunit of Ragulator [13, 15, 18]
Summary
To identify the interacting partners of ARL5B, we employed a nanobody-based GFP-binding protein (GBP) pull-down assay [23, 24]. We tested whether HSC70 binds to ARL5B in a nucleotidedependent manner We performed a His pull-down assay where His-ARL5B protein was immobilized on Ni-NTA beads and incubated with MDA-MB-231 cell extracts in the presence of 5 mM ADP or 5 mM ATP. The result from the binding assay showed that ARL5B interacted with HSC70 preferentially in the presence of ADP compared with ATP (Fig. 1E). To delineate the importance of the N-terminal helix of ARL5B in binding to HSC70, we deleted the first 15 residues from the N-terminal of ARL5B, denoted as [ΔN15]ARL5B, and purified the His-tagged [ΔN15]ARL5B protein (Fig. S2A) We did a His pull-down assay by immobilizing His-ARL5B and His-[ΔN15]ARL5B on Ni-NTA beads and incubating with MDA-MB-231 cell extracts. Since it was known that mutating Q alone in the KFERQ-like sequence was sufficient to perturb the
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