Abstract
Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.
Highlights
The generation of functional transport carriers at the transGolgi network (TGN)4 requires the coordinated recruitment of coat proteins, membrane deformation, vesicle formation, and vesicle fission (1–3)
Because constitutive secretion of matrix metalloproteinase (MMP) from the TGN were predominantly affected by PKD2, we focused on this isoform in our subsequent experiments
Two major protein families have been identified as regulators of vesicle biogenesis and fission controlling secretion at the TGN: the protein kinase D (PKD) family and adenosyl-ribosylation factor family of small G-proteins (ARFs)-GTPases
Summary
The generation of functional transport carriers at the transGolgi network (TGN)4 requires the coordinated recruitment of coat proteins, membrane deformation, vesicle formation, and vesicle fission (1–3). To investigate the role of ARL1 and PKD2 in Arfaptin2-TGN localization we performed FRAP experiments in HeLa cells to monitor Arfaptin2-YFP recovery after its depletion at the TGN in the presence of different interaction partners (Fig. 3, A–C). In addition to the FRAP experiments, we performed quantitative co-localization studies with Arfaptin2-YFP in HeLa cells at the TGN following depletion of ARL1 or PKD2, respectively.
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