Laurel Thomas

Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface.

Furin is a membrane‐associated endoprotease that efficiently cleaves precursor proteins on the C‐terminal side of the consensus sequence, Arg‐X‐Lys/Arg‐Arg1, and has been proposed to catalyze these reactions in both exocytic and endocytic compartments. To study its biosynthesis and routing, a furin construct (designated fur/f) containing the FLAG epitope tag inserted on the C‐terminal side of the enzymes autoproteolytic maturation site was used. Introduction of the epitope tag had no effect on the expression, proteolytic maturation or activity of furin. Analysis of the localization of fur/f by immunofluorescence microscopy showed that its staining pattern largely overlapped with those of several Golgi‐associated markers. Treatment of cells with brefeldin A caused the fur/f distribution to collapse around the microtubule organizing center, indicating that furin is concentrated in the trans‐Golgi network (TGN). Immunoelectron microscopy showed unequivocally that furin resides in the TGN where it colocalized with TGN38. In agreement with its proposed activity in multiple compartments, antibody uptake studies showed that fur/f cycles between the cell surface and TGN. Furthermore, targeting to the TGN requires sequences in the cytoplasmic tail of the enzyme. Pulse‐chase and immunofluorescence analyses demonstrated that proregion removal occurs in the endoplasmic reticulum and that cleavage may be required for exist from this compartment. Finally, we show that proregion removal is necessary but not sufficient for enzyme activation.

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.

PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization

We report the role of one member of a novel gene family, PACS-1, in the localization of trans-Golgi network (TGN) membrane proteins. PACS-1 directs the TGN localization of furin by binding to the proteases phosphorylated cytosolic domain. Antisense studies show TGN localization of furin and mannose-6-phosphate receptor, but not TGN46, is strictly dependent on PACS-1. Analyses in vitro and in vivo show PACS-1 has properties of a coat protein and connects furin to components of the clathrin-sorting machinery. Cell-free assays indicate TGN localization of furin is directed by a PACS-1-mediated retrieval step. Together, these findings explain a mechanism by which membrane proteins in mammalian cells are localized to the TGN.

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.

PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic

PACS‐1 is a cytosolic protein involved in controlling the correct subcellular localization of integral membrane proteins that contain acidic cluster sorting motifs, such as furin and human immunodeficiency virus type 1 (HIV‐1) Nef. We have now investigated the interaction of PACS‐1 with heterotetrameric adaptor complexes. PACS‐1 associates with both AP‐1 and AP‐3, but not AP‐2, and forms a ternary complex between furin and AP‐1. A short sequence within PACS‐1 that is essential for binding to AP‐1 has been identified. Mutation of this motif yielded a dominant‐negative PACS‐1 molecule that can still bind to acidic cluster motifs on cargo proteins but not to adaptor complexes. Expression of dominant‐negative PACS‐1 causes a mislocalization of both furin and mannose 6‐phosphate receptor from the trans‐Golgi network, but has no effect on the localization of proteins that do not contain acidic cluster sorting motifs. Furthermore, expression of dominant‐negative PACS‐1 inhibits the ability of HIV‐1 Nef to downregulate MHC‐I. These studies demonstrate the requirement for PACS‐1 interactions with adaptor proteins in multiple processes, including secretory granule biogenesis and HIV‐1 pathogenesis.

Interaction of furin in immature secretory granules from neuroendocrine cells with the AP‐1 adaptor complex is modulated by casein kinase II phosphorylation

The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans‐Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP‐1 type adaptor complex and clathrin‐coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin‐coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP‐1, a component of the TGN/ISG‐localized clathrin sorting machinery. Interaction between AP‐1 and furin is dependent on phosphorylation of the enzymes cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation‐dependent association of furin with AP‐1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT‐20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.

The PHD/LAP-Domain Protein M153R of Myxomavirus Is a Ubiquitin Ligase That Induces the Rapid Internalization and Lysosomal Destruction of CD4

ABSTRACT The genomes of several poxviruses contain open reading frames with homology to the K3 and K5 genes of Kaposis sarcoma-associated herpesvirus (KSHV) and the K3 gene of murine gammaherpesvirus 68, which target major histocompatibility complex class I (MHC-I) as well as costimulatory molecules for proteasomal or lysosomal degradation. The homologous gene product of myxomavirus (MV), M153R, was recently shown to reduce the cell surface expression of MHC-I. In addition, normal MHC-I surface expression was observed in cells infected with MV lacking M153R (J. L. Guerin, J. Gelfi, S. Boullier, M. Delverdier, F. A. Bellanger, S. Bertagnoli, I. Drexler, G. Sutter, and F. Messud-Petit, J. Virol. 76:2912-2923, 2002). Here, we show that M153R also downregulates the T-cell coreceptor CD4 and we study the molecular mechanism by which M153R achieves the downregulation of CD4 and MHC-I. Upon M153R expression, CD4 was rapidly internalized and degraded in lysosomes, whereas deletion of M153R from the genome of MV restored CD4 expression. The downregulation of both CD4 and MHC-I was dependent on the presence of lysine residues in their cytoplasmic tails. Increased ubiquitination of CD4 was observed upon coexpression with M153R in the presence of inhibitors of lysosomal acidification. Surface expression of CD4 was restored upon overexpression of Hrs, a ubiquitin interaction motif-containing protein that sorts ubiquitinated proteins into endosomes. Moreover, the purified PHD/LAP zinc finger of M153R catalyzed the formation of multiubiquitin adducts in vitro. Our data suggest that M153R acts as a membrane-bound ubiquitin ligase that conjugates ubiquitin to the cytoplasmic domain of substrate glycoproteins, with ubiquitin serving as a lysosomal targeting signal. Since a similar mechanism was recently proposed for KSHV K5, it seems that members of the unrelated families of gamma-2 herpesviruses and poxviruses share a common immune evasion mechanism that targets host cell immune receptors.

Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg156-Asn-Lys-Arg159-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg↓) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for ≈20% of all known XLHED cases, with mutations occurring most frequently at Arg156, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg156Asn-Lys-Arg159↓- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor α1-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.

HIV-1 Nef Binds PACS-2 to Assemble a Multikinase Cascade That Triggers Major Histocompatibility Complex Class I (MHC-I) Down-regulation ANALYSIS USING SHORT INTERFERING RNA AND KNOCK-OUT MICE

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE65 and PXXP75. The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met20. How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE65-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP75 to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2-/- mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.

A PACS‐1, GGA3 and CK2 complex regulates CI‐MPR trafficking

The cation‐independent mannose‐6‐phosphate receptor (CI‐MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans‐Golgi network (TGN) to lysosomes. Cycling of CI‐MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS‐1, which directs endosome‐to‐TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS‐1 bind to an overlapping CI‐MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2‐activated phosphorylation cascade controlling PACS‐1‐ and GGA3‐mediated CI‐MPR sorting. PACS‐1 links GGA3 to CK2, forming a multimeric complex required for CI‐MPR sorting. PACS‐1‐bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI‐MPR and early endosomes. Bound CK2 also phosphorylates PACS‐1Ser278, promoting binding of PACS‐1 to CI‐MPR to retrieve the receptor to the TGN. Our results identify a CK2‐controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system.