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.

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 A(1) (PA-PLA(1)). 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-PLA(1) family (PA-PLA(1) 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-PLA(1). 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.

Lewy bodies (LBs) are the pathologic hallmark of Parkinson’s 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.

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.

NVL (nuclear VCP-like protein), a member of the AAA-ATPase family, is known to exist in two forms with N-terminal extensions of different lengths in mammalian cells. Here, we show that they are localized differently in the nucleus; NVL2, the major species, is mainly present in the nucleolus, whereas NVL1 is nucleoplasmic. Mutational analysis demonstrated the presence of two nuclear localization signals in NVL2, one of which is shared with NVL1. In addition, a nucleolar localization signal was found to exist in the N-terminal extra region of NVL2. The nucleolar localization signal is critical for interaction with ribosomal protein L5, which was identified as a specific interaction partner of NVL2 on yeast two-hybrid screening. The interaction of NVL2 with L5 is ATP-dependent and likely contributes to the nucleolar translocation of NVL2. The physiological implication of this interaction was suggested by the finding that a dominant negative NVL2 mutant inhibits ribosome biosynthesis, which is known to take place in the nucleolus.

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 Mg(2+)-ATP treatment in the presence of NSF and alpha-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.

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 alpha-SNAP, an adaptor that serves as a link between the chaperone ATPase NSF and SNAREs. This predicts that alpha-SNAP may suppress apoptosis by competing with antiapoptotic proteins for the BH3 domain of BNIP1. Indeed, overexpression of alpha-SNAP markedly delayed staurosporine-induced apoptosis. Our results shed light on possible crosstalk between apparently independent cellular events, apoptosis and ER membrane fusion.

Gaf-1/Rip11 encoded by the clone KIAA0857 participates in endosomal recycling through the interaction with both γ-SNAP, a member of the soluble NSF attachment protein family, and a small GTPase, Rab11. Gaf-1/Rip11 and other Rab11-interacting proteins constitute a novel protein family that is involved in the endocytic pathways. Here we report the presence of an alternative splice variant of Gaf-1/Rip11 named Gaf-1b. Gaf-1b also interacts with γ-SNAP and is expressed ubiquitously in tissues except for liver. Subcellular fractionation analysis revealed that Gaf-1b, as well as Gaf-1/Rip11, is mainly present in the microsomal fraction. Overexpression of Gaf-1b, like that of Gaf-1/Rip11, affected the morphology of recycling endosomes. These results suggest that Gaf-1b has a similar function to Gaf-1/Rip11.

VCP/p97 is involved in a variety of cellular processes, including membrane fusion and ubiquitin-dependent protein degradation. It has been suggested that adaptor proteins such as p47 and Ufd1p confer functional versatility to VCP/p97. To identify novel adaptors, we searched for proteins that interact specifically with VCP/p97 by using the yeast two-hybrid system, and discovered a novel VCP/p97-interacting protein named small VCP/p97-interacting protein (SVIP). Rat SVIP is a 76-amino acid protein that contains two putative coiled-coil regions, and potential myristoylation and palmitoylation sites at the N terminus. Binding experiments revealed that the N-terminal coiled-coil region of SVIP, and the N-terminal and subsequent ATP-binding regions (ND1 domain) of VCP/p97, interact with each other. SVIP and previously identified adaptors p47 and ufd1p interact with VCP/p97 in a mutually exclusive manner. Overexpression of full-length SVIP or a truncated mutant did not markedly affect the structure of the Golgi apparatus, but caused extensive cell vacuolation reminiscent of that seen upon the expression of VCP/p97 mutants or polyglutamine proteins in neuronal cells. The vacuoles seemed to be derived from endoplasmic reticulum membranes. These results together suggest that SVIP is a novel VCP/p97 adaptor whose function is related to the integrity of the endoplasmic reticulum.

gamma-Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (gamma-SNAP) is capable of stabilizing a 20 S complex consisting of NSF, alpha-SNAP, and SNAP receptors (SNAREs), but its function in vesicular transport is not fully understood. Our two-hybrid analysis revealed that gamma-SNAP, unlike alpha-SNAP, interacts directly with NSF, as well as Gaf-1/Rip11, but not with SNAREs. Gaf-1/Rip11 is a gamma-SNAP-associated factor that belongs to the Rab11-interacting protein family. To gain insight into the molecular basis for the interactions of gamma-SNAP with NSF and Gaf-1/Rip11, we determined the regions of the three proteins involved in protein-protein interactions. gamma-SNAP bound to NSF via its extreme C-terminal region, and the full-length NSF was needed to interact with gamma-SNAP. Both the N-terminal and C-terminal regions of gamma-SNAP were required for the binding to Gaf-1/Rip11. Gaf-1/Rip11 bound to gamma-SNAP via its C-terminal domain comprising a putative coiled-coil region. Although the C-terminal domain of Gaf-1/Rip11 also interacts with Rab11, the binding of gamma-SNAP and Rab11 to Gaf-1/Rip11 was not mutually exclusive. Rather, Gaf-1/Rip11 was capable of serving a link between gamma-SNAP and Rab11. A complex comprising gamma-SNAP and Gaf-1/Rip11 was disassembled in a process coupled to NSF-mediated ATP hydrolysis, suggesting that the interaction between gamma-SNAP and Gaf-1/Rip11 is of functional significance.

p125, a mammalian Sec23p-interacting protein, exhibits sequence homology with bovine testis phosphatidic acid-preferring phospholipase A(1). In this study, we identified and characterized a new homologue of p125, KIAA0725p. KIAA0725p exhibited remarkable sequence similarity with p125 throughout the entire sequence determined but lacked an N-terminal proline-rich, Sec23p-interacting region. In vitro binding analysis showed that KIAA0725p does not bind to Sec23p. KIAA0725p possessed phospholipase A(1) activity preferentially for phosphatidic acid. We examined the effects of overexpression of KIAA0725p on the morphology of organelles. Overexpression of KIAA0725p, like that of p125, caused dispersion of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins located in the Golgi region and caused aggregation of the endoplasmic reticulum. Our results indicate that KIAA0725p is a new member of the phosphatidic acid-preferring phospholipase A(1) protein family and suggest that the cellular function of KIAA0725p is different from that of p125.

We showed previously that overexpression of the α subunit of Gz or Gi2 suppresses nordihydroguaiaretic acid-induced Golgi disassembly. To determine whether the active form of Gα is required to maintain the structure of the Golgi apparatus, we examined the effects of a series of Gα GAPs, regulators of G protein signaling (RGS)proteins, on the Golgi structure. Expression of RGSZ1 or RGSZ2, both of which exhibit high selectivity for Gαz, markedly induced dispersal of the Golgi apparatus, whereas expression of RGS proteins that are rather selective for Gαq or other Gαi species did not. A mutated RGSZ1, which is deficient in the interaction with Gαz, did not induce Golgi disassembly. These results suggest that the active form of Gαz, but not Gαi2,is crucial for maintenance of the structure of the Golgi apparatus. Consistent with this idea, Golgi disruption also took place in cells transfected with a dominant-negative Gαz mutant. Although previous studies showed that the expression of Gαz is confined to neuronal cells and platelets, immunofluorescence and mRNA expression analyses revealed that it is also expressed, albeit at low levels, in non-neuronal cells, and is located in the Golgi apparatus. These results taken together suggest a general regulatory role for Gαz in the control of the Golgi structure.

PACE4A is a member of the mammalian subtilisin-like proprotein convertase family which is responsible for the proteolytic activation of precursors into their biologically active forms. Previously we reported that the maturation of proPACE4A occurs via a intramolecular autoactivation and cleavage of the propeptide is a rate-limiting step for the secretion of PACE4A (Nagahama et al., FEBS Lett. (1998) 434, 155–159). Although PACE4A is a putative secretory enzyme, it matures and is secreted much slower than general secretory proteins. In this study, we investigated the molecular mechanism underlying this slow maturation. The deletion of 25 amino acids at the carboxy terminus is sufficient for a marked acceleration in both the maturation and secretion of PACE4A. The carboxyl-truncated proPACE4A existed only as a monomer-sized form in the endoplasmic reticulum, whereas the wild type of proPACE4A existed in larger forms. Further, the fusion construct of yellow fluorescent protein and the carboxy-terminal sequence of PACE4A associated with the proPACE4A moiety and inhibited maturation. Thus the carboxy terminus of PACE4A functions as a potent autoinhibitor of its activation, resulting in the retention of proPACE4A in the endoplasmic reticulum. These findings indicate that PACE4A activity is highly controlled by a unique system at post-translational level.

Disassembly of the Golgi apparatus is elicited by the action of nordihydroguaiaretic acid (NDGA) and this disassembly is prevented by the activation of heterotrimeric G proteins. In the present study we showed that overexpression of Gαz or Gαi2 significantly suppresses the disassembly of the Golgi apparatus induced by NDGA. Overexpression of Gβ1γ2, on the other hand, had no effect on NDGA‐induced Golgi disassembly. Gαz neither blocked Golgi disassembly induced by brefeldin A or nocodazole, nor interfered with protein transport, suggesting its specificity on the action of NDGA. Our results suggest that the α subunits of heterotrimeric G proteins are responsible for the maintenance of the Golgi structure.

N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg(2+)-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg(2+)-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle.

We examined the effects of short chain and long chain ceramides on the stability of the Golgi apparatus. Short chain ceramides, C(2)- and C(6)-ceramides, blocked brefeldin A-induced Golgi disassembly without affecting the rapid release of Golgi coat proteins, whereas they did not inhibit brefeldin A-induced tubulation of endosomes. Both short chain ceramides also retarded Golgi disassembly induced by nordihydroguaiaretic acid and nocodazole, suggesting that they stabilize the Golgi apparatus. In contrast to short chain ceramides, natural long chain ceramides, when incorporated into cells or formed within cells upon treatment with sphingomyelinase or metabolic inhibitors, enhanced brefeldin A-induced Golgi disassembly. These results suggest that sphingolipid metabolism is implicated in the stability of the Golgi apparatus.

Serum albumin is synthesized as a larger precursor form, proalbumin, which undergoes proteolytic processing at a dibasic site by a hepatic proprotein convertase within the secretory pathway to generate the mature form. Although furin, a member of the subtilisin-like proprotein convertase (SPC) family, was thought to be the only candidate hepatic convertase for proalbumin, SPC family members other than furin were recently suggested to also be involved in proalbumin processing. This study was designed to identify the endogenous proprotein convertases involved in proalbumin processing. Since human hepatoma HepG2 cells are highly differentiated and produce major plasma proteins, this cell line was used as a model for hepatocytes. Northern blot analysis revealed that PACE4, furin and PC8 of the SPC family were expressed in HepG2 cells as well as in the liver. Ribonuclease protection assay showed that PACE4A-II mRNA is the major transcript in HepG2 cells among the PACE4 isoforms. The coexpression studies showed that furin, PACE4A-II and PC8 were all able to convert proalbumin to albumin correctly. To elucidate the roles of these endogenous SPC family members in proalbumin processing, the antisense RNA for PACE4, furin and PC8 was stably expressed in HepG2 cells, respectively. The expression of each antisense RNA resulted in approximately 30% inhibition of endogenous proalbumin processing. We therefore concluded that PACE4 and PC8, as well as furin, are involved in the processing of proalbumin in HepG2 cells, and that these SPC family members are functionally redundant in this processing.

Subtilisin-like proprotein convertases (SPCs) are a family of proteases that are responsible for the processing of many precursors at sites containing multiple basic residues. The region-specific expression of SPCs during the early development of the brain implicates this class of proteases in the functional plasticity of the nervous system. L1 is a Ca(2+)-independent neural cell adhesion molecule that may play a critical role in some aspects of axonal guidance. L1 has several molecular forms including 200, 140, and 80 kDa (L1-200, L1-140, and L1-80). L1-140/80 are thought to be generated by proteolysis of L1-200 at its extracellular region, which contains the multiple basic recognition motif for SPCs, To demonstrate the involvement of SPCs in this process, we have coexpressed L1 with the SPCs in HEK293 cells. Results demonstrated that SPC1 (furin) is most active at cleaving L1-200 with additional cleavage of the generated products. SPC6 (PC6) and SPC7 (PC8/LPC) are less active than SPC1; however, L1-200 was correctly processed into L1-140/80. These results suggest that SPCs are responsible for the proteolytic processing of L1 observed in the developing brain and thus play a regulatory role by modulating biological activity of L1 during brain development and neural regeneration.

PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui et al. reported that the ectopic expression of alpha1-antitrypsin variant Portland (alpha1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y. et al. (1998) EMBO J. 17, 4735-4743]. TGFbeta-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to alpha1-PDX, have been reported for PACE4. In this study, we examined whether alpha1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that alpha1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An alpha1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease(s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca(2+)-dependent protease with an optimal Ca(2+) requirement of 2 mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by alpha1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by alpha1-PDX causes abnormal embryogenic development.

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.