Masami Nagahama

Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi

ZW10, a dynamitin‐interacting protein associated with kinetochores, is known to participate directly in turning off of the spindle checkpoint. In the present study, we show that ZW10 is located in the endoplasmic reticulum as well as in the cytosol during interphase, and forms a subcomplex with RINT‐1 (Rad50‐interacting protein) and p31 in a large complex comprising syntaxin 18, an endoplasmic reticulum‐localized t‐SNARE implicated in membrane trafficking. Like conventional syntaxin‐binding proteins, ZW10, RINT‐1 and p31 dissociated from syntaxin 18 upon Mg2+‐ATP treatment in the presence of NSF and α‐SNAP, whereas the subcomplex was not disassembled. Overexpression, microinjection and knockdown experiments revealed that ZW10 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. The present results disclose an unexpected role for a spindle checkpoint protein, ZW10, during interphase.

Involvement of BNIP1 in apoptosis and endoplasmic reticulum membrane fusion

BNIP1, a member of the BH3‐only protein family, was first discovered as one of the proteins that are capable of interacting with the antiapoptotic adenovirus E1B 19‐kDa protein. Here we disclose a totally unexpected finding that BNIP1 is a component of the complex comprising syntaxin 18, an endoplasmic reticulum (ER)‐located soluble N‐ethylmaleimide‐sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE). Functional analysis revealed that BNIP1 participates in the formation of the ER network structure, but not in membrane trafficking between the ER and Golgi. Notably, a highly conserved leucine residue in the BH3 domain of BNIP1 plays an important role not only in the induction of apoptosis but also in the binding of α‐SNAP, an adaptor that serves as a link between the chaperone ATPase NSF and SNAREs. This predicts that α‐SNAP may suppress apoptosis by competing with antiapoptotic proteins for the BH3 domain of BNIP1. Indeed, overexpression of α‐SNAP markedly delayed staurosporine‐induced apoptosis. Our results shed light on possible crosstalk between apparently independent cellular events, apoptosis and ER membrane fusion.

p125 Is Localized in Endoplasmic Reticulum Exit Sites and Involved in Their Organization

Transport vesicles coated with the COPII complex, which is assembled from Sar1p, Sec23p-Sec24p, and Sec13p-Sec31p, are involved in protein export from the endoplasmic reticulum (ER). We previously identified and characterized a novel Sec23p-interacting protein, p125, that is only expressed in mammals and exhibits sequence homology with phosphatidic acid-preferring phospholipase A1 (PA-PLA1). In this study, we examined the localization and function of p125 in detail. By using immunofluorescence and electron microscopy, we found that p125 is principally localized in ER exit sites where COPII-coated vesicles are produced. Analyses of chimeric proteins comprising p125 and two other members of the mammalian PA-PLA1 family (PA-PLA1 and KIAA0725p) showed that, for localization to ER exit sites, the p125-specific N-terminal region is critical, and the putative lipase domain is interchangeable with KIAA0725p but not with PA-PLA1. RNA interference-mediated depletion of p125 affected the organization of ER exit sites. The structure of the cis-Golgi compartment was also substantially disturbed, whereas the medial-Golgi was not. Protein export from the ER occurred without a significant delay in p125-depleted cells. Our study suggests that p125 is a mammalian-specific component of ER exit sites and participates in the organization of this compartment.

Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation.

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3δ from association with gp78 and p97/VCP, which is accompanied by decreases in CD3δ ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3δ and misfolded Z variant of α-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.

Functional characterization of Asp-317 mutant of human renin expressed in COS cells

Renin is an unique aspartyl (acid) protease with optimal activity at neutral pH. It has been suggested that Ala‐317 of human renin contributes to neutral optimum pH of the enzyme [(1984) FEBS Lett. 174, 102–111]. The hypothesis was verified by the characterization of mutant renin in which Ala‐317 was replaced with Asp by a site‐directed mutagenesis. Wild‐type and mutant renins, which were expressed in COS cells, exhibited different pH‐activity profiles and optimum pH of the mutant enzyme was lower than that of the wild‐type enzyme. This result suggests that Ala‐317 of human renin plays an important role in the determination of optimum pH of the enzyme.

A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4

SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an alpha1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.

Biosynthetic processing and quaternary interactions of proprotein convertase SPC4 (PACE4)

SPC4 (PACE4), a member of the eukaryotic family of subtilisin‐like proprotein convertases, is synthesized as a proenzyme (proSPC4) which undergoes proteolytic removal of N‐terminal propeptide during transit through the secretory pathway. As this propeptide processing seems to be a key event in the functional expression of SPC4, we have investigated its mechanism and the intracellular site where it occurs. In transfected fibroblast cells, the 110‐kDa proSPC4 undergoes slow cleavage to generate a 103‐kDa mature enzyme in the endoplasmic reticulum (ER). Site‐directed mutagenesis studies demonstrate that the proteolytic activation of SPC4 occurs mainly through a unimolecular autocatalytic process and propeptide cleavage is a prerequisite for its export from the ER. Sedimentation velocity and chemical cross‐linking analysis demonstrate that the precursor protein in the cells exists as both a monomer and a dimer‐sized complex whereas mature SPC4 exists only as a monomer. These results suggest that the cleavage of the N‐terminal propeptide of SPC4 plays a regulatory role in its activation and secretion through the change in its oligomeric state.

Dominant-negative effect of mutant valosin-containing protein in aggresome formation

Lewy bodies (LBs) are the pathologic hallmark of Parkinsons disease. Recent studies revealed that LBs exhibit several morphologic and molecular similarities to aggresomes. Aggresomes are perinuclear aggregates representing intracellular deposits of misfolded proteins. Recently, valosin‐containing protein (VCP) was one of the components of LBs, suggesting its involvement in LB formation. Here, we showed the localization of VCP in aggresomes induced by a proteasome inhibitor in cultured cells. Cells overexpressing mutant VCP (K524M: D2) showed reduced aggresome formation relative to those overexpressing wild‐type and mutant (K251M: D1) VCPs. Our findings suggest that the D2 domain is involved in aggresome formation.

UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation

AAA ATPase VCP and its yeast ortholog Cdc48, in a complex with the Ufd1-Npl4 heterodimer as an adaptor, play an essential role in endoplasmic reticulum-associated degradation (ERAD). Several UBX domain-containing proteins function to recruit ubiquitylated substrates to VCP/Cdc48 by binding both VCP/Cdc48 and other ERAD components such as ubiquitin ligases. Here we show that mammalian UBXD1 is an additional UBX domain-containing protein involved in the ERAD process. UBXD1 is a cytosolic protein that interacts with VCP and Derlin-1. Overexpression of UBXD1 in cells causes selective dissociation of Ufd1 from VCP, resulting in inhibition of mutant cystic fibrosis transmembrane conductance regulator (CFTR) degradation by ERAD. Additionally, depletion of endogenous UBXD1 protein by RNA interference also results in a defect in CFTR degradation. Collectively, these findings suggest that UBXD1 is a regulatory component of ERAD that may modulate the adaptor binding to VCP.

Subtilisin-like proprotein convertase PACE4 is required for skeletal muscle differentiation.

Most growth factors stimulate myoblast proliferation and prevent differentiation, whereas insulin-like growth factors (IGFs) promote myoblast differentiation through the phosphatidylinositol 3-kinase (PI3K) pathway. Subtilisin-like proprotein convertases (SPCs) are involved in cell growth and differentiation via activation of pro-growth factors. However, the role of SPCs in myogenesis remains poorly understood. Here we show that PACE4, a member of the SPC family, plays a critical role in myogenic differentiation of C2C12 cells. PACE4 mRNA levels increased markedly during myogenesis, whereas the expression of other member of SPC family, furin and PC6, remained unchanged. The expression pattern of pro-IGF-II, which is processed extracellularly by SPCs, was similar to that of PACE4. The expression of shRNA targeting PACE4, but not furin, suppressed the expression of the muscle-specific myosin light chain (MLC). Interestingly, reduced expression of MLC was restored following treatment with recombinant mature IGF-II. Finally, we demonstrated that the PI3K inhibitor LY294002 blocked the induction of PACE4 mRNA, a result not observed when another myogenic differentiation inhibitor, SB203580 (p38 MAP kinase inhibitor), was employed, indicating the presence of a positive feedback loop regulating PACE4 expression. These results suggest that PACE4 plays an important role in myogenic differentiation through its association with the IGF-II pathway.