Identification of a pH sensor in the furin propeptide that regulates enzyme activation.

The folding and activation of furin occur through two pH- and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg107/ generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site 69HRGVTKR75/, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg107. Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2- and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg75 to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is a model for other proteins.

PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis.

The endoplasmic reticulum (ER) and mitochondria form contacts that support communication between these two organelles, including synthesis and transfer of lipids, and the exchange of calcium, which regulates ER chaperones, mitochondrial ATP production, and apoptosis. Despite the fundamental roles for ER-mitochondria contacts, little is known about the molecules that regulate them. Here we report the identification of a multifunctional sorting protein, PACS-2, that integrates ER-mitochondria communication, ER homeostasis, and apoptosis. PACS-2 controls the apposition of mitochondria with the ER, as depletion of PACS-2 causes BAP31-dependent mitochondria fragmentation and uncoupling from the ER. PACS-2 also controls formation of ER lipid-synthesizing centers found on mitochondria-associated membranes and ER homeostasis. However, in response to apoptotic inducers, PACS-2 translocates Bid to mitochondria, which initiates a sequence of events including the formation of mitochondrial truncated Bid, the release of cytochrome c, and the activation of caspase-3, thereby causing cell death. Together, our results identify PACS-2 as a novel sorting protein that links the ER-mitochondria axis to ER homeostasis and the control of cell fate, and provide new insights into Bid action.

HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway.

The HIV-1 Nef-mediated downregulation of cell surface MHC-I molecules to the trans-Golgi network (TGN) enables HIV-1 to escape immune surveillance. However, the cellular pathway used by Nef to downregulate MHC-I is unknown. Here, we show that Nef and PACS-1 combine to usurp the ARF6 endocytic pathway by a PI3K-dependent process and downregulate cell surface MHC-I to the TGN. This mechanism requires the hierarchical actions of three Nef motifs-the acidic cluster 62EEEE(65), the SH3 domain binding site 72PXXP(75), and M(20)-in controlling PACS-1-dependent sorting to the TGN, ARF6 activation, and sequestering internalized MHC-I to the TGN, respectively. These data provide new insights into the cellular basis of HIV-1 immunoevasion.