Yukio Ikehara

The GM130 and GRASP65 Golgi proteins cycle through and define a subdomain of the intermediate compartment

Integrating the pleomorphic membranes of the intermediate compartment (IC) into the array of Golgi cisternae is a crucial step in membrane transport, but it is poorly understood. To gain insight into this step, we investigated the dynamics by which cis-Golgi matrix proteins such as GM130 and GRASP65 associate with, and incorporate, incoming IC elements. We found that GM130 and GRASP65 cycle via membranous tubules between the Golgi complex and a constellation of mobile structures that we call late IC stations. These stations are intermediate between the IC and the cis-Golgi in terms of composition, and they receive cargo from earlier IC elements and deliver it to the Golgi complex. Late IC elements are transient in nature and sensitive to fixatives; they are seen in only a fraction of fixed cells, whereas they are always visible in living cells. Finally, late IC stations undergo homotypic fusion and establish tubular connections between themselves and the Golgi. Overall, these features indicate that late IC stations mediate the transition between IC elements and the cis-Golgi face.

Immunolocalization of tissue non-specific alkaline phosphatase in mice

Abstract Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-μm tissue slices, and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP. Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes.

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).

Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes

The effects of brefeldin A on intracellular transport and posttranslational modification of complement C3 (C3) were studied in primary culture of rat hepatocytes. In the control culture C3 was synthesized as a pre‐cursor (pro‐C3), which was processed to the mature form with α‐ and β‐subunits before its discharge into the medium. In the presence of brefeldin A the secretion of C3 was strongly blocked, resulting in accumulation of pro‐C3. However, after a prolonged interval the mature form of C3 was finally secreted. The results indicate that brefeldin A impedes translocation of pro‐C3 to the Golgi complex where pro‐C3 is converted to the mature form, but not its proteolytic processing, in contrast to the effects of monensin and weakly basic amines.

The highly conserved DAD1 protein involved in apoptosis is required for N-linked glycosylation

Background: The tsBN7 cell line is one of the temperature‐sensitive mutants for cell proliferation which have been isolated from the BHK21 cell line derived from the golden hamster. It has a mutation in the DAD1 gene encoding a 12.5 kDa highly conserved protein through evolution, and enters apoptosis at the restrictive temperature due to this mutation.

Active production and membrane anchoring of carcinoembryonic antigen observed in normal colon mucosa

Normal colon mucosa was found to produce carcinoembryonic antigen (CEA) quite actively as cancerous tissues do when maintained in an organ culture, although the fresh normal mucosae contained a very small quantity of CEA unlike cancerous tissues. This is consistent with an active expression of CEA mRNA in normal mucosa comparable to that in cancerous tissues actively producing CEA, and suggests that the normal cell product was rapidly released into the lumen of digestive tract and turned into normal fecal antigens (NFAs) previously found in feces. 3H-Labeled precursors of glycophospholipid such as ethanolamine and stearic acid were incorporated into CEA produced by both normal and cancerous tissues, suggesting that CEA in normal mucosa is anchored to the cell membrane through a glycophospholipid as in cancerous tissues.

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.

Bafilomycin A1 inhibits the targeting of lysosomal acid hydrolases in cultured hepatocytes

Effects of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase, on the synthesis and processing of cathepsin D and cathepsin H were investigated in primary cultured rat hepatocytes. Pulse-chase experiments showed that after being synthesized as procathepsin D and procathepsin H the precursors were converted into mature forms in the control cells as the chase time elapsed. However, in the presence of 5 x 10(-7) M of bafilomycin A1, both precursors were largely secreted into the medium and no mature forms were found within the cells. Thus bafilomycin A1 mimics lysosomotropic amines with regard to perturbation of the targeting of lysosomal acid hydrolases. In contrast, bafilomycin A1 was found not to inhibit processings of proalbumin and procomplement component 3, which are thought to occur at the acidic trans-Golgi, implying that the proteolytic event of the proproteins is not sensitive to an increase of intra-Golgi pH. The results suggest that bafilomycin A1 is useful as a pH-perturbant to study the role of acidity in living cells.

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.

Monensin inhibits the conversion of proalbumin to serum albumin in cultured rat hepatocytes

Abstract Effects of monensin, a carboxylic ionophore, on intracellular transport of albumin were studied in primary cultured rat hepatocytes. The lag time after which newly synthesized albumin first appeared in medium was 10 min in the control cells, while it was prolonged to 40 min in the monensin-treated cells. In addition, this inhibition of secretion by monensin was accompanied by an intracellular accumulation of proalbumin. The results strongly suggest that monensin arrests the intracellular transport of proalbumin before the site where its conversion takes place.