Kiyotaka Hatsuzawa

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.

p125 Is a Novel Mammalian Sec23p-interacting Protein with Structural Similarity to Phospholipid-modifying Proteins

COPII-coated vesicles are involved in protein transport from the endoplasmic reticulum to the Golgi apparatus. COPII consists of three parts: Sar1p and the two protein complexes, Sec23p-Sec24p and Sec13p-Sec31p. Using a glutathioneS-transferase fusion protein with mouse Sec23p, we identified a novel mammalian Sec23p-interacting protein, p125, which is clearly distinct from Sec24p. The N-terminal region of p125 is rich in proline residues, and the central and C-terminal regions exhibit significant homology to phospholipid-modifying proteins, especially phosphatidic acid preferring-phospholipase A1. We transiently expressed p125 and mouse Sec23p in mammalian cells and examined their interaction. The results showed that the N-terminal region of p125 is important for the interaction with Sec23p. We confirmed the interaction between the two proteins by a yeast two-hybrid assay. Overexpression of p125, like that of mammalian Sec23p, caused disorganization of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus, suggesting its role in the early secretory pathway.

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to α-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.

Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum

The quality of cargo proteins in the endoplasmic reticulum (ER) is affected by their motion during folding. To understand how the diffusion of secretory cargo proteins is regulated in the ER, we directly analyze the motion of a single cargo molecule using fluorescence imaging/fluctuation analyses. We find that the addition of two N-glycans onto the cargo dramatically alters their diffusion by transient binding to membrane components that are confined by hyperosmolarity. Via simultaneous observation of a single cargo and ER exit sites (ERESs), we could exclude ERESs as the binding sites. Remarkably, actin cytoskeleton was required for the transient binding. These results provide a molecular basis for hypertonicity-induced immobilization of cargo, which is dependent on glycosylation at multiple sites but not the completion of proper folding. We propose that diffusion of secretory glycoproteins in the ER lumen is controlled from the cytoplasm to reduce the chances of aggregation.

Proteolytic cleavages of proalbumin and complement Pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease☆

We have recently purified and characterized a truncated soluble form of furin from which the predicted transmembrane domain and cytoplasmic tail were deleted (Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) J. Biol. Chem. 267, 16094-16099). Our results showed that furin resembles the yeast Kex2 protease with respect to both its enzymic properties and substrate specificity. Here we demonstrate that the soluble form of furin is capable of converting the precursors of albumin and the third component of complement (proalbumin and pro-C3, respectively) in vitro to mature proteins. Thus furin mimics the Ca(2+)-dependent proalbumin and pro-C3 convertases found in the Golgi membranes (Brennan, S. O., and Peach, R. J. (1988) FEBS Lett. 229, 167-170; Oda, K. (1992) J. Biol. Chem. 267, 17465-17471). Furthermore we show that the variant alpha 1-antitrypsin Pittsburgh, which is a specific inhibitor of the Golgi proalbumin convertase, inhibits not only the Golgi pro-C3 convertase, but also the soluble furin. These results suggest a role for furin in the cleavage of proproteins transported via the constitutive pathway.

Regulation of the Golgi structure by the α subunits of heterotrimeric G proteins

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.

Hypothetical protein KIAA0079 is a mammalian homologue of yeast Sec24p

The Sec23p‐Sec24p complex is a component of COPII‐coated vesicles that mediate protein transport from the endoplasmic reticulum in yeast. The mammalian hypothetical protein KIAA0079 (KIAA0079p) exhibits sequence similarity to yeast Sec24p. KIAA0079p was co‐eluted with mammalian Sec23p on gel filtration. In vitro binding experiments revealed that the C‐terminal region of KIAA0079p binds to the N‐terminal region of mammalian Sec23p. Overexpression of KIAA0079p caused a defect in protein export from the endoplasmic reticulum. These results support the idea that KIAA0079p is a functional homologue of yeast Sec24p.

NSF is required for the brefeldin A-promoted disassembly of the Golgi apparatus

N‐Ethylmaleimide‐sensitive factor (NSF) is required for multiple pathways of vesicle‐mediated protein transport. Microinjection of a monoclonal anti‐NSF antibody almost completely blocked brefeldin A‐promoted Golgi disassembly without affecting the rapid release of β‐COP, a subunit of the Golgi coat proteins (COPI), from the Golgi apparatus. Similar results were obtained using a dominant‐negative NSF which is known to compete with endogenous NSF. The present results suggest that an NSF‐mediated step is present in the brefeldin A‐promoted disassembly of the Golgi apparatus.

Gaf-1b is an alternative splice variant of Gaf-1/Rip11

Gaf-1/Rip11 encoded by the clone KIAA0857 participates in endosomal recycling through the interaction with both gamma-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 gamma-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.

The protein complexes involved in membrane fusion

Using a microlens attached Nipkow disk scanner-confocal microscope, we observed bovine adrenal chromafIin cells after staining with a fluorescent dye, quinacrine. Upon exocytosis of a chromafftn vesicle, the fluorescence of the vesicle rapidly decreased. A significant number of vesicles maintained a dark fluorescence even after this response. The dark fluorescence, derived from residual quinacrine in the granule, was distinguished from a fluorescence of a quinacrine-filled vesicle moved from the focal plane. After partial release of quinacrine, some vesicles underwent a I?111 exocytotic release, and others detached from the membrane to move into the deep part of the cytoplasm without showing the full exocytosis (kiss and run). These findings suggested the presence of a new recycling pathway for vesicles after exocytosis, and provided evidence against the widely accepted hypothesis of quanta1 release