Miwa Sohda

The interaction of two tethering factors, p115 and COG complex, is required for Golgi integrity.

The vesicle‐tethering protein p115 functions in endoplasmic reticulum–Golgi trafficking. We explored the function of homologous region 2 (HR2) of the p115 head domain that is highly homologous with the yeast counterpart, Uso1p. By expression of p115 mutants in p115 knockdown (KD) cells, we found that deletion of HR2 caused an irregular assembly of the Golgi, which consisted of a cluster of mini‐stacked Golgi fragments, and gathered around microtubule‐organizing center in a microtubule‐dependent manner. Protein interaction analyses revealed that p115 HR2 interacted with Cog2, a subunit of the conserved oligomeric Golgi (COG) complex that is known another putative cis‐Golgi vesicle‐tethering factor. The interaction between p115 and Cog2 was found to be essential for Golgi ribbon reformation after the disruption of the ribbon by p115 KD or brefeldin A treatment and recovery by re‐expression of p115 or drug wash out, respectively. The interaction occurred only in interphase cells and not in mitotic cells. These results strongly suggested that p115 plays an important role in the biogenesis and maintenance of the Golgi by interacting with the COG complex on the cis‐Golgi in vesicular trafficking.

Interaction of Golgin-84 with the COG Complex Mediates the Intra-Golgi Retrograde Transport

The coiled‐coil Golgi membrane protein golgin‐84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin‐84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin‐84 as the tether for COPI vesicles of intra‐Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin‐84 knockdown (KD) cells. The depletion of golgin‐84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra‐Golgi soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and cis‐Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex‐dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin‐84. Surprisingly, the interaction between golgin‐84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin‐84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin‐84 with COG plays an important role in the tethering process of intra‐Golgi retrograde vesicle traffic.

Identification and Characterization of GCP16, a Novel Acylated Golgi Protein That Interacts with GCP170

GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH2-terminal region (positions 137–237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [3H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys69 and Cys72, accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface.

Intense correlation between protein-conjugated acrolein and primary Sjögren's syndrome

BACKGROUND We recently found that an increased plasma concentration of protein-conjugated acrolein is a good biomarker for stroke. Therefore we determine whether the concentration of protein-conjugated acrolein is increased in saliva from patients with primary Sjögrens syndrome. METHODS Stimulated whole-mixed saliva was collected from 10 patients and 13 control subjects. The concentration of protein-conjugated acrolein in saliva and plasma was measured by either Western blotting or enzyme-linked immunosorbent assay. RESULTS The concentration of protein-conjugated acrolein, especially albumin-conjugated acrolein, was greatly increased in saliva from patients with primary Sjögrens syndrome (p<0.001). The concentration of protein-conjugated acrolein was inversely correlated with the flow rate of saliva. CONCLUSION The results indicate that the concentration of protein-conjugated acrolein, a marker of cell or tissue damage, in saliva is well correlated with seriousness of primary Sjögrens syndrome.

Association of nonsense mutation in GABRG2 with abnormal trafficking of GABAA receptors in severe epilepsy

Mutations in GABRG2, which encodes the γ2 subunit of GABAA receptors, can cause both genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Most GABRG2 truncating mutations associated with Dravet syndrome result in premature termination codons (PTCs) and are stably translated into mutant proteins with potential dominant-negative effects. This study involved search for mutations in candidate genes for Dravet syndrome, namely SCN1A, 2A, 1B, 2B, GABRA1, B2, and G2. A heterozygous nonsense mutation (c.118C>T, p.Q40X) in GABRG2 was identified in dizygotic twin girls with Dravet syndrome and their apparently healthy father. Electrophysiological studies with the reconstituted GABAA receptors in HEK cells showed reduced GABA-induced currents when mutated γ2 DNA was cotransfected with wild-type α1 and β2 subunits. In this case, immunohistochemistry using antibodies to the α1 and γ2 subunits of GABAA receptor showed granular staining in the soma. In addition, microinjection of mutated γ2 subunit cDNA into HEK cells severely inhibited intracellular trafficking of GABAA receptor subunits α1 and β2, and retention of these proteins in the endoplasmic reticulum. The mutated γ2 subunit-expressing neurons also showed impaired axonal transport of the α1 and β2 subunits. Our findings suggested that different phenotypes of epilepsy, e.g., GEFS+ and Dravet syndrome (which share similar abnormalities in causative genes) are likely due to impaired axonal transport associated with the dominant-negative effects of GABRG2.

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure.

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF4- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.

Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406. Structural importance of the crown domain.

Hypophosphatasia, a congenital metabolic disease related to the tissue‐nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS‐1 cells and in stable CHO‐K1 Tet‐On cells. Compared with the wild‐type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild‐type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol‐anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild‐type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.

Localization of the PP2A B56γ Regulatory Subunit at the Golgi Complex: Possible Role in Vesicle Transport and Migration

The BL6 subline was derived from the F10 line, which was derived from the B16 mouse melanoma cell line. BL6 cells are more invasive than F10 cells and differ genetically from F10 cells by an alteration of the gene encoding the B56gamma regulatory subunit of protein phosphatase 2A (PP2A). This alteration results in the transcription of mRNA encoding a truncated variant of the B56gamma1 isoform (Deltagamma1). When F10 cells were stained with a polyclonal antibody that recognizes three B56gamma isoforms, B56gamma1, B56gamma2, and B56gamma3, the immunofluorescent signals co-localized well with the cis-Golgi marker proteins. When BL6 cells were fractionated in a sucrose gradient, B56gamma1 and B56gamma2, but not B56gamma3, were present in the Golgi-enriched fraction. This fraction also contained the catalytic subunit of PP2A. FLAG-tagged Deltagamma1 preferentially localized to the trans-Golgi area rather than the cis-Golgi. This localization was the same as that of FLAG-tagged B56gamma1. NIH3T3 cells stably expressing Deltagamma1 transported a mutant viral protein from the endoplasmic reticulum to the plasma membrane much faster than wild-type cells. Their directional migration, as assessed by the advance of cells into a cell-free area, was also elevated. As Deltagamma1 reduces the activity of the B56gamma-containing PP2A holoenzymes, these results suggest that the normal holoenzymes suppress vesicle transport and that Deltagamma1 might increase the invasive ability of BL6 cells by activating Golgi function.

Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids

When expressed in COS cells, human prorenin was secreted into the medium without being processed to an active renin. Co-expression of furin, a mammalian homologue of the yeast KEX2 gene product, did not affect proteolytic processing of prorenin. A mutant proreninR-4 constructed by site-directed mutagenesis of Pro (-4) to Arg was not cleaved by an endoprotease in the COS cell. However, proreninR-4 was detectably cleaved to yield the active renin upon co-transfection with furin DNA, indicating that Arg at position -4 is important for recognition and processing by furin in addition to the absolute requirement for paired basic amino acids. Another mutant precursor in which Leu (+1) of proreninR-4 was replaced with Ser was found to be much more efficiently processed than proreninR-4, regardless of co-expression of furin. The results suggest that not only a basic amino acid at position -4 but also Leu at position +1 significantly affect the processing of prorenin catalyzed by the COS cell endoprotease or furin.

Intracellular processing of complement pro-C3 and proalbumin is inhibited by rat α1-protease inhibitor variant (Met352→Arg) in transfected cells

Complement C3, when its cDNA was transfected into COS-1 cells, was synthesized as a precursor, pro-C3, which was intracellularly processed into the alpha and beta subunits, although not completely. A cDNA for rat alpha 1-protease inhibitor (alpha 1-PI) was mutated in vitro to encode its variant with the modified active site (Met352----Arg). In cells co-transfected with the mutant alpha 1-PI cDNA and the C3 cDNA, pro-C3 expressed was secreted without being processed into the subunits. Co-transfection of the mutant alpha 1-PI cDNA and the albumin cDNA also resulted in the inhibition of intracellular conversion of proalbumin into serum-type albumin. No inhibition of the processing of each preform was observed in cells co-transfected with the normal alpha 1-PI cDNA. Taken together, the results indicate that the alpha 1-PI variant (Met352----Arg) expressed inhibits specifically an intracellular enzyme which is involved in the proteolytic processing of both pro-C3 and proalbumin.