Tokiro Ishikawa

EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step

All three mammalian EDEM family members possess mannosidase activity and are necessary for glycoprotein degradation, but EDEM2 performs a unique, rate-limiting, first mannose trimming step upstream of EDEM1 and EDEM3.

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

The endoplasmic reticulum (ER) membrane-bound transcription factors ATF6α and ATF6β mediate adjustment of chaperone levels to increased demands in the ER, which is essential for development of the notochord; the latter synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

Gamma-ray irradiation promotes premature meiosis of spontaneously differentiating testis–ova in the testis of p53-deficient medaka (Oryzias latipes)

In this study, the roles of p53 in impaired spermatogenic male germ cells of p53-deficient medaka were investigated by analyzing histological changes, and gene expressions of 42Sp50, Oct 4 and vitellogenin (VTG2) by RT-PCR or in situ hybridization in the testes. We found that a small number of oocyte-like cells (testis–ova) differentiated spontaneously in the cysts of type A and early type B spermatogonia in the p53-deficient testes, in contrast to the wild-type (wt) testes in which testis–ova were never found. Furthermore, ionizing radiation (IR) irradiation increased the number of testis–ova in p53-deficient testes, increased testis–ova size and proceeded up to the zygotene or pachytene stages of premature meiosis within 14 days after irradiation. However, 28 days after irradiation, almost all the testis–ova were eliminated presumably by p53-independent apoptosis, and spermatogenesis was restored completely. In the wt testis, IR never induced testis–ova differentiation. This is the first study to demonstrate the pivotal role of the p53 gene in the elimination of spontaneous testis–ova in testes, and that p53 is not indispensable for the restoration of spermatogenesis in the impaired testes in which cell cycle regulation is disturbed by IR irradiation.

Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway

Higher eukaryotes, but not yeast, are able to extract severely misfolded glycoproteins from the endoplasmic reticulum–associated degradation (ERAD) pathway for glycoproteins and target them to the ERAD pathway for non-glycoproteins to maintain the homeostasis of the ER.

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage–specific manner

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.

Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish.

SEL1L-dependent substrates require Derlin2/3 and Herp1/2 for endoplasmic reticulum-associated degradation

Accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ATF6 pathway is one of the three major pathways in vertebrates. Although ATF6, a transmembrane-type glycoprotein in the ER, functions as a UPR sensor/transducer, it is an unstable protein with a half-life of approximately 2 h and is constitutively subjected to the ER-associated degradation system with the location of the misfolded part in the ER lumen (ERAD-L). ERAD-L substrates are delivered to the cytosol through the retrotranslocon, which likely contains HRD1 (E3), gp78 (E3), SEL1L (a partner of HRD1), Derlin1/2/3 and Herp1/2. We previously showed that ATF6 represents a novel transmembrane-type ERAD-L substrate requiring both EDEM1/2/3-mediated mannose trimming and SEL1L. Here, by constructing and analyzing chicken DT40 cells deficient in various components of the retrotranslocon, we show that degradation of ATF6 requires Derlin2 or Derlin3 and that Derlin2 and Derlin3 are redundant for ERAD-L of ATF6. We further show that degradation of ATF6 requires Herp1 or Herp2 and that Herp1 and Herp2 are redundant for ERAD-L of ATF6. Furthermore, by investigating five more ERAD-L substrates, we show that SEL1L-dependent substrates require Derlin2/3 and Herp1/2 regardless of their soluble or transmembrane nature. Our results suggest that ERAD-L substrates take several routes to the cytosol. The HRD1-engaged route 1 requires SEL1L, Derlin2 or Derlin3, and Herp1 or Herp2, whereas the HRD1-engaged route 2 does not require them functionally. It remains to be determined whether the latter requires Derlin1 and whether these two routes are compositionally distinct.Key words: endoplasmic reticulum, proteasome, protein degradation, protein misfolding, ubiquitin.

A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase

Genetic analysis is facilitated by the efficient production of transgenic strains expressing a DNA of interest as a single copy at a designated chromosomal location. However, technical progress toward this goal in medaka fish (Oryzias latipes), a vertebrate model organism, has been slow. It is well known that phiC31 integrase enables efficient site-directed transgenesis by catalyzing the recombination of an attP DNA motif in a host genome with an attB motif in a targeting vector. This system was pioneered in medaka using the Sleeping Beauty transposon system, and the attP site was established at three chromosomal locations. However, this number appeared insufficient with regard to genetic linkage between the attP-landing site and a genetically modified locus of interest. Here, to establish a collection of transgenic strains of medaka, we introduced an attP motif into the medaka genome using the Ac/Ds maize transposon system and established 12 independent transgenic strains harboring a single copy of the attP motif in at least 11 of the 24 medaka chromosomes. We designed an attB-targeting vector that was integrated efficiently and precisely into the attP-landing site, and with which the DNA of interest was efficiently transmitted to germline cells. Extraneous sequences in the integrants derived from the bacterial backbone of the attB-targeting vector as well as a transgenic fluorescence marker present in the attP-landing site were removable through flippase-mediated recombination. Further, an advanced targeting vector with a heart-specific recombination marker served as a useful tool for easily screening phiC31 integrase-mediated recombinant G0 embryos, leading to the efficient establishment of transgenic strains. Thus, our resources advance genetic research in medaka.