Journal of Insect Biotechnology and Sericology 86, 105-112 (2017)

The physiological accumulation of mutant fibroin light chains induces an unfolded response in the posterior gland of the cocoon Nd-sD strain of silkworm Bombyx mori

Tadashi Takahashi1, Masao Miyazaki1, Shin-ichiro Kidou2, Yoshiki Matsui1, Ying An1, Taku Ozaki3, Koichi Suzuki1＊＊ and Tetsuro Yamashita1＊

1 Faculty of Agriculture, Iwate University, Ueda, Morioka, Iwate 020-8550, Japan 2 Graduate School of Natural Sciences, Nagoya City University, Mizuho-cho, Mizuho-ku, Nagoya 467-8501, Japan 3 Faculty of Science and Engineering, Iwate University, Ueda, Morioka, Iwate 020-8550, Japan (Received May 15, 2017; Accepted July 4, 2017)

Fibroin, a major component of silk fiber, is composed of light (L) chains, heavy chains, and fhx/P25 in the silk- worm Bombyx mori. Sericin cocoon (Nd-sD) is a silkworm strain expressing a mutant fibroin L chain that is accu- mulated in the endoplasmic reticulum (ER) of posterior silk glands (PSGs). However, little is known about the effects of accumulation in PSGs in Nd-sD strain. We compared the PSG gene expression profiles of 5th-instar larvae of Nd-sD strain and the fibroin-producing normal strain. cDNA representational difference analysis identi- fied candidate genes whose expression levels were higher in Nd-sD strain than in normal strain. We focused on heat shock and cathepsin B, a major lysosomal protease. We confirmed upregulation of BiP(GRP78), Hsp20.8, and cathepsin B at the transcriptional and/or translational levels. These results suggest that the accu- mulation of mutant L chain induces the unfolded protein response in PSGs of Nd-sD strain, which will be a valu- able tool for protein quality-control studies of silk grand. Key words: Bombyx mori, fibroin, unfolded protein response, molecular chaperone

ponents (Inoue et al., 2000). The fibroin L chain, fibroin INTRODUCTION H chain, and fhx/P25 are assembled in the endoplasmic Fibroin is the major silk protein component synthesized reticulum (ER) in a 6:6:1 molecular ratio of L chain:H in the posterior silk gland (PSG) of the silkworm Bombyx chain:fhx/P25. A previous study demonstrated that the (H- mori. Fibroin is composed of three proteins: 30-kD light L)6 fhx/P25 complex structure is essential for the secre- (L) chain (Yamaguchi et al., 1989), 350-kD heavy (H) tion of fibroin into the PSG lumen; therefore, it is called chain (Zhou et al., 2000), and 25-kD fibrohexamerin (fhx)/ the “fibroin elementary unit” (Inoue et al., 2004). P25 (Chevillard et al., 1986). Intact fibroin L chain is The silkworm mutant, Sericin cocoon (Nd-sD), has fi- composed of 262 residues, the first 18 of broin secretion levels that are less than 0.3% of those of which are cleaved as a signal peptide and the subsequent fibroin-producing strains (Takei et al., 1987). Therefore, N-terminal is acetylated (Yamaguchi et al., 1989). this fibroin-secretion -deficient mutant produces a very thin, The deduced fibroin L-chain polypeptide contains three naked-pupa cocoon that consists mostly of sericin (Fig. 1A). Cys residues. An intramolecular disulfide bond is formed Genetic analyses revealed that Nd-sD strain has a deletion between Cys83 and Cys142 in the fibroin L chain. Cys172 of approximately 10 kbp in the Fib-L gene (Fig. 1B). Since forms an intermolecular disulfide linkage to Cys-c20 (20th the deleted region contains four exons (IV, V, VI, and VII) residues from the carboxyl terminus) of the fibroin H of the fibroin L chain, the resulting gene has two new ex- chain (Tanaka et al., 1999). The middle portion of the ons (IV′ and V′). The L-chain polypeptide of the Nd-sD amino acid sequence of the fibroin H chain contains- re mutant is a hybrid amino acid sequence in which the first peated Gly-Ala sequences that form a highly hydrophobic 105 residues are identical to the native fibroin L-chain crystalline domain. Fhx/P25 is a glycoprotein that pos- polypeptide, but the remaining sequence containing Cys172 sesses three high mannose-type Asn-linked oligosaccha- is completely different from the original (Mori et al., ride chains that interact with the H chain, which inhibits 1995). Therefore, the mutant fibroin L chain cannot form non-specific interactions and aggregations of fibroin com- a disulfide bond with the fibroin H chain. The lack of this  disulfide bond causes the production of misfolded fibroin; *To whom correspondence should be addressed. i.e., a secretion-deficient phenotype. A previous study- ob D Fax &Tel: +81-19-621-6157. served the enlarged ER in the PSG of the Nd-s mutant Email: [email protected] using an electron microscope and found that fibroin com- **Present address: Biococoon Laboratories Inc., Research and De- ponents accumulated in the ER (Gamo and Sato, 1985). velopment Center by Collaboration of Morioka city and Iwate Uni- Consequently, the deficiency in the fibroin elementary unit versity, Morioka, Iwate 020-8551, Japan because of mutant fibroin L-chain production results in 106 Takahashi et al.

Fig. 1. (A) Fifth instar larvae and cocoons of DAIZO and Nd-sD strain. (B) Genetic map of normal fib-L and Nd-sD strain fib-L genes. the fibroin secretion-deficient phenotype in Nd-sD strain. strain. We compared the gene expression profiles in the Based on these findings, we hypothesized that the- pro PSGs of days-2-6 5th-instar larvae of normal strain, a fi- duction of mutant fibroin L chain is involved in quality broin-producing silkworm strain, and Nd-sD strain using control of proteins in the PSG. Molecular chaperones pro- cDNA representational difference analysis (RDA). RDA is vide the machinery for protein quality control in cells a PCR-based subtractive hybridization method to clarify (Buchberger et al., 2010). They discriminate between na- the differences between two DNA samples (Hubank and tive and misfolded proteins or protein complexes after Schatz, 1994). Because we found that genes including which they can induce the folding of unfolded proteins heat shock proteins were more highly expressed in Nd-sD into their appropriate folded states. If they find mis- or strain than in normal strain, we also compared protein ex- unfolded proteins in the ER, the intracellular trafficking pression levels between the strains. for such proteins is inhibited. Mis- and unfolded proteins that were already transported to the Golgi apparatus are MATERIALS AND METHODS returned to the ER for restoration. Further expression and accumulation of mis- and unfolded proteins leads to the Silkworms unfolded protein response (UPR) (Mori, 2000). The UPR The Sericin cocoon (Nd-sD) mutant strain was gifted by consists of four stages: suppression of translation, induc- the Insect Genetics Laboratory of the National Institute of tion of chaperone genes, activation of ER-associated deg- Agrobiological Sciences, Japan. The silkworm Bombyx radation (ERAD), and finally induction of apoptosis. We mori DAIZO strain, whose secretion level of fibroin is therefore suggest that the accumulation of mutant fibroin normal, was supplied by the Laboratory of Applied Ento- components in the PSG of the Nd-sD mutant strain leads mological Science of Iwate University, Japan. These strains to the UPR in the ER. were maintained by feeding artificial foods under a 12-h The aim of the present study was to examine the effect light/dark photoperiod at 25°C. Fifth-instar larvae of both of the expression of the mutant fibroin L chain on the strains were used in this study. quality control of proteins in the PSG of the Nd-sD mutant Unfolded protein response of silk gland 107

Table 1. Primer pairs used for RT-PCR Target gene Forward primer Reverse primer Fibroin L chain (normal) 5’-GGATCCGTCATTAACTCTTACACAG-3’ 5’-GAATTCTTAGACGTGAACCTGGCTGG-3’ Fibroin L chain (Nd-sD) 5’-GGATCCGT ATGGGTTCGGTTATTTCG-3’ 5’-GAATTCCTATTCACACATT CGTTTAT-3’ fhx/P25 5’-GTCTAGCTGTAGCCGCTGTG-3’ 5’-C TGCGCTGTGCGTTGGTCAT-3’ Cathepsin B 5’-GGATCCATGTTTATTTCGCG TGCGGCGTAT-3’ 5’-GTCGACCTAGTGTTCATCTAAGAACG GTTC-3’ Hsc 70-4 5’-GAATTCATGGCAAAAGCACCCGCAGT-3’ 5’-GTCGACAAGTGTGGTGTGGAATGTTG-3’ BiP 5’-GTCGACATGGT CAAGATGCGGTGGAG-3’ 5’-GTCGACCATCTTCAAGCGTCTTCTTCT -3’ Hsp 20.8 5’-GGATCCATGTCTCTTCTACCATTC GTG-3’ 5’-GAATTCACCCTACTTATTTTCGGCAG-3’

Representational difference analysis (RDA) of for 8 h with probes for BiP, Hsp20.8, Hsc70-4, or cathep- cDNA sin B, which were obtained using RT-PCR and labeled us- A modified method established by Hubank and Schatz ing an AlkPhos Direct Labeling Module (GE Healthcare). (Hubank and Schatz, 1994) was used for RDA of cDNA. The primers for these genes are summarized in Table 1. Total RNAs were extracted from PSGs of the DAIZO and The membranes were incubated with CDP-Star detection Nd-sD strains at appropriate stages using the TRIzol re- reagent (GE Healthcare). agent (Invitrogen, Carlsbad, CA, USA). cDNA of each sample was synthesized from 1 μg of total RNA using the Western blotting cDNA Synthesis Kit (M-MLV version, Takara). After di- PSGs were prepared from days-2-6 5th-instar larvae of gesting the synthesized cDNA with Sau3AI, two linker DAIZO and Nd-sD strains, and lysed in RIPA buffer [50 mM primers, RBA1 (5’-AGCACTCTCCAGCCTCTCACCG Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1% DOC, 1% AG-3’) and RBA2 (5’-GATCCTCGGTGA-3’), were ligat- SDS, 1 mM EDTA, 1× Complete Mini Protease Inhibitor ed on both ends of the cDNA. RBA-linked cDNA frag- Cocktail (Roche, Mannheim, Germany)]. The fibroin aggre- ments were amplified by PCR using Ex Taq (Takara) and gates were precipitated by the centrifugation at 15,000 × g, RBA1. PCR products were digested with Sau3AI and ex- and the supernatant (20-μg aliquot) was separated by cess primers were removed. The same protocol was ap- SDS-PAGE using a 10% polyacrylamide gel with under plied to substitute the RBA linkers for the NBA linkers, reducing conditions. Proteins in the gel were electroblotted NBA1 (5’-AGGCAACTGTGCTATCCGAGGGAG-3’) and onto PVDF membrane (Millipore, Bedford, MA, USA) NBA2 (5’-GATCCTCCCTCG-3’), when preparing NBA- and detected by western blotting using antisera, following linked cDNA fragments. We mixed 100 ng of NBA-linked a previous study (Tamura et al., 2002) Rabbit antisera for cDNA fragments (objective sample from Nd-sD strain) and BiP, cathepsin B, and Hsc70-4 were raised as described 5 μg of RBA-linked cDNA fragments (control sample from previously (Tamura et al., 2002). Antigen polypeptide se- normal strain) to hybridize them at 67°C for 16 h in 5 μL quences were as follows: Fibroin L-chain, acetyl-SVTIN of the hybridization buffer (25 mM EPPS, 2.5 mM EDTA, QYSDNEIPRDIDDGKC; BiP, CLYQGQGGVPPPGAP 1 M NaCl, pH 8.25). The hybridization solution was used EDDDFKDEL; cathepsin B, CGIESSIVTGEPFLDEH; for PCR using Ex Taq, and NBA1. The PCR product was Hsc70-4, CGGAAPGAGGAGPTIEEVD; Hsp20.8, CGP treated with mung bean nuclease at 37°C for 30 min and VRKEIKDQSEGTQDAENK. Anti-fhx/P25 antiserum was then used as template for PCR with the NBA1. Finally, provided by Dr. Hiromitsu Tanaka at the National Institute cDNA fragments that were separated in a 2% agarose gel of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. were purified with a MinElute Gel Extraction Kit (Qiagen), and ligated into pCRII-TOPO vectors (Invitrogen). Such RESULTS vectors were transformed into Escherichia coli (TOP10), and colonies were randomly picked to sequence the in- Higher expression genes in Nd-sD strain than in serted cDNA. The internal sequences were determined DAIZO strain using an ABI 3100 DNA sequencer (GE Healthcare) and Our study started with exhaustive screening of candi- analyzed using the program BlastX (http://www.ncbi.nlm. date genes whose expression levels were higher in Nd-sD nih.gov/BLAST/). strain than in DAIZO strain. We conducted RDA to com- pare gene expression of PSGs in Nd-sD and DAIZO strains Northern blotting using 5th-instar larvae, the life-history stage at which fi- Total RNA (20-μg aliquot) was separated on a 1% aga- broin-producing strains synthesize and secrete fibroin ele- rose-MOPS gel and blotted onto a Hybond-N+ membrane mentary units at their highest levels. Table 2 shows the (GE Healthcare). The membrane was hybridized at 55°C candidate genes that were more highly expressed in Nd-sD 108 Takahashi et al.

Table 2. RDA screening of candidate genes whose expression levels may have been higher in Nd-sD strain than in DAIZO 3 Day/5th-instar Nd-sD strain > DAIZO Accession no. Gene Number of clones AAR10100 Similar to Drosophila melanogaster qm [Drosophila yakuba] 1 BAA34444 KIAA0724 protein [Homo sapiens] 1 BAA76303 Gag-like protein [Bombyx mori] 2 BAB21762 Gag-like protein [Bombyx mori] 1 BAC57926 Reverse transcriptase [Bombyx mori] 6 CAA81531 Mutant L-chain [Bombyx mori] 8 NP_001027625 Tensin homologue [Ciona intestinalis] 1 XP_394756 PREDICTED: similar to ENSANGP00000021019 [Apis mellifera] 1 XP_550566 Putative glycine-rich protein [Oryza sativa (japonica cultivar-group)] 1 XP_759834 Hypothetical protein UM03687.1 [Ustilago maydis 521] 1 YP_336421 Putative zinc-binding dehydrogenase [Burkholderia pseudomallei 1710b] 1

4 Day/5th-instar Nd-sD strain > DAIZO Accession no. Gene Number of clones AAH81044 MGC81780 protein [Xenopus laevis] 1 AAV34862 Ribosomal protein S6 [Bombyx mori] 1 BAA32395 BiP (GRP78) [Bombyx mori] 2 BAC57926 Reverse transcriptase [Bombyx mori] 2

6 Day/5th-instar Nd-sD strain > DAIZO Accession no. Gene Number of clones BAA32395 BiP (GRP78) [Bombyx mori] 1 BAB40804 Cathepsin B [Bombyx mori] 1 BAB92074 HSc70.4 [Bombyx mori] 8 CAA81531 Mutant fibroin L-chain [Bombyx mori] 4 CAE69817 Hypothetical protein CBG16133 [Caenorhabditis briggsae] 1 strain, while Table 3 shows those more highly expressed BiP and Hsp20.8 expression levels in Nd-sD strain in DAIZO. In the tester RDA using cDNA of PSGs ob- In this study, we focused on two heat shock proteins, tained from day-2 and day-5 5th-instar larvae of Nd-sD BiP (GRP78) and Hsc70-4, as candidate genes whose lev- strain, no fragment was identified by final PCR. Although els may have been higher in Nd-sD strain than in DAIZO. these tables contained many putative genes, we found that Genes of the three heat shock proteins were screened only the levels of mutant fibroin L chain may have been higher in Nd-sD strain by RDA. BiP mRNA levels were markedly in Nd-sD strain than in DAIZO. By contrast, levels of higher in Nd-sD strain than in DAIZO, especially from fibroin L chain and fhx/P25 may have been higher in day-5 5th-instar larvae (Fig. 3A). High expression of BiP DAIZO than in Nd-sD strain. Western blotting using anti was also confirmed at the translational level (Fig. 3B). By fibroin L-chain antibodies prepared from regions that were contrast, Hsc70-4 was expressed at similar levels during conserved between the original and mutant fibroin L all stages of 5th-instar larvae in both Nd-sD and DAIZO chains confirmed that normal fibroin 28 kD L chain was strains at transcriptional and translational levels. Based on expressed at higher levels in DAIZO than in Nd-sD strain, the result, we also examined small heat shock proteins, whereas 31 kD mutant fibroin L chain was detectable only such as Hsc20.8, in these strains. Interestingly, Hsc20.8 in Nd-sD strain (Fig. 2A). In western blotting using anti- level was higher in Nd-sD strain than in DAIZO at stages fhx/P25 antibodies, fhx/P25 levels were higher in DAIZO of 5th and 6th -instar larvae. than in Nd-sD strain (Fig. 2B). These results demonstrated that RDA works for finding genes whose expression lev- Cathepsin B expression levels in Nd-sD strain els differ between the DAIZO and Nd-sD strains. Additionally, we focused on cathepsin B, which belongs to a family of lysosomal cysteine proteases and plays an Unfolded protein response of silk gland 109

Table 3. RDA screening of candidate genes whose expression levels may have been higher in DAIZO than in Nd-sD strain 4 Day/5th-instar DAIZO > Nd-sD strain Accession no. Gene Number of clones AAF48263 CG1640-PB, isoform B [Drosophila melanogaster] 1 BAA19776 Pol protein [Bombyx mori] 1 BAA76303 Gag-like protein [Bombyx mori] 2 BAC06462 reverse transcriptase [Papilio xuthus] 1 BAC57926 reverse transcriptase [Bombyx mori] 1 XP_394120 PREDICTED: similar to abdominal-A protein [Apis mellifera] 1

5 Day/5th-instar DAIZO > Nd-sD strain Accession no. Gene Number of clones BBA76303 Gag-like protein [Bombyx mori] 3 BAC57926 reverse transcriptase [Bombyx mori] 1 CAA35180 silk fibroin light chain [Bombyx mori] 1

6 Day/5th-instar DAIZO > Nd-sD strain Accession no. Gene Number of clones A33470 fibroin light chain precursor - silkworm 1 AAL83649 silk fibroin [Bombyx mori] 1 AAL83698 translation elongation factor 2 [Spodoptera exigua] 1 AAV34812 ribosomal protein L3 [Bombyx mori] 1 BAB39500 fibroin P25 [Bombyx mandarina] 3 CAA27804 P25 [Bombyx mori] 1 important role in intracellular proteolysis (Authier et al., teins that increased in the ER of PSGs in Nd-sD strain due 1996). Cathepsin B was screened as a candidate whose to the accumulation of mutant fibroin L chain. levels may have been higher in Nd-sD strain than in DAIZO The RDA of cDNA in PSGs between Nd-sD and DAIZO in day-6 5th-instar larvae. Cathepsin B mRNA was mark- strains suggested that expression levels of molecular chap- edly upregulated in Nd-sD strain day-3 5th-instar larvae erones, such as BiP and Hsc70-4 were increased in Nd-sD (Fig. 4). Western blotting using anti-cathepsin B antibodies strain in addition to the mutant fibroin L chain. We con- detected a 31-kD band in DAIZO, as well as 35- and 37- firmed that BiP, but not Hsc70-4, were increased in PGSs kD bands corresponding to inactive premature cathepsin B of Nd-sD strain during the 5th instar at both the transcrip- in Nd-sD strain. tional and translational levels. By contrast, fhx/P25, a ma- jor component of the fibroin elementary unit, decreased in PGSs of Nd-sD strain, possibly because the expression of DISCUSSION fhx/P25 is suppressed by the first stage of UPR, i.e. sup- In this study, we compared gene expression levels of pression of mRNA translation, in Nd-sD strain. PSGs between Nd-sD and DAIZO strains (Fig. 5). Although BiP is an ER molecular chaperone involved in the qual- DAIZO, which produces normal cocoons, is not the wild- ity control of de novo synthesized proteins and is particu- type strain of Nd-sD, we used it because the wild type was larly important for correct protein folding in the ER unavailable. Therefore, we used not only Nd-sD strain (Kleizen and Braakman, 2004). BiP reversibly interacts cDNA but also DAIZO cDNA as testers for RDA, and with hydrophobic regions of unfolded proteins and enhanc- found that normal fibroin L chain was expressed at higher es the stability of the binding proteins (Gething, 1999). levels in DAIZO than in Nd-sD strain, whereas mutant fi- This mechanism is important for inducing the UPR in broin L chain was detectable only in Nd-sD strain. A small cells (Rutkowski and Kaufman, 2004). Therefore, the up- amount of normal fibroin L chain detected in Nd-sD strain regulation of BiP in PSGs of the Nd-sD strain during the was thought to result from the homologous recombination 5th instar may play an essential role in stabilizing misfold- with normal fibroin L chain gene in the process of the ed fibroin elementary units caused by the mutant fibroin L maintaining the mutant strain. We believe that RDA be- chain. Such a cellular response using BiP may be impor- tween DAIZO and Nd-sD strains allowed us to find pro- tant to prevent the formation of irreversible large protein 110 Takahashi et al.

Fig. 2. Expression levels of fibroin light (L) chains and fhx/P25 in day-2-6 5th-instar larvae of DAIZO and Nd-sD silk- worm strains. Proteins (20-μg aliquot) isolated from posterior silk glands (PSGs) of DAIZO and Nd-sD silkworm strains were analyzed by western blotting using (A) anti-fibroin L-chain and (B) fhx/P25 antibodies.

Fig. 3. Expression levels of BiP, Hsc70.4, and Hsp20.8 in day-2-6 5th-instar larvae of DAIZO and Nd-sD silkworm strains. (A) Total RNAs (20-μg aliquot) isolated from posterior silk glands (PSGs) of DAIZO and Nd-sD strains were an- alyzed by northern blotting using BiP, Hsc70.4, and Hsp20.8 DNA probes. Amounts and qualities of RNA samples were confirmed by 28s rRNAs. (B) Proteins (20-μg aliquot) isolated from PSGs of DAIZO and Nd-sD strains were ana- lyzed by western blotting using anti-BiP and Hsc70.4 antibodies.

Fig. 4. Expression levels of cathepsin B in day-2-6 5th-instar larvae of DAIZO and Nd-sD silkworm strains. (A) Total RNAs (20-μg aliquot) isolated from posterior silk glands (PSGs) of DAIZO and Nd-sD strains were analyzed by north- ern blotting using a cathepsin B DNA probe. (B) Proteins (20-μg aliquot) isolated from PSGs of DAIZO and Nd-sD strains were analyzed by western blotting using anti-cathepsin B antibodies. aggregates of the misfolded fibroin elementary units, which of cell stresses in the cytoplasm (de Jong et al., 1993). The may be toxic to PSG cells. major function of these proteins is to bind unfolded poly- Hsp20.8 expression in PGSs of the Nd-sD strain was peptides to protect cells from aggregated proteins. There- upregulated only on days 5 and 6 of the 5th instar. These fore, we suggest that misfolded fibroin elementary units proteins are categorized as small heat shock proteins with accumulated in the ER may be transferred to the cytosol alpha-crystallin domains, which are induced by a variety via retro-translocon channels of the ER membrane, then Unfolded protein response of silk gland 111

Fig. 5. Schematic model of the protein transport and unfolded protein responses in posterior silk gland cells of DAIZO and Nd-sD strains. sHsps: small Heat shock proteins. stabilized by Hsp20.8, and finally degraded by the ubiqui- (QEILK) between residues 63 and 67 in its N-terminus tin- system. (Majeski and Dice, 2004). This suggests that mutant fibro- Cathepsin B expression was also upregulated in PGSs in L chains not used to produce fibroin elementary units of the Nd-sD strain on day 3 of the 5th instar. Since ca- are degraded by the CMA pathway. thepsin B is a major lysosomal protease, we suggest that Gene transfer and editing technologies, including the the upregulation of cathepsin B indicates the activation of CRISPR/Cas9 system, enable us to develop transgenic the lysosome in PSGs of the Nd-sD strain. Their PGSs silkworms that make recombinant proteins by using the contain not only the active form of 31-kD cathepsin B but system that produces silk-gland proteins (Iizuka et al., also immature inactive forms of 35- and 37-kD cathepsin 2009; Inoue et al., 2005; Tamura et al., 2000). To im- B in Nd-sD strains. It was reported that de novo synthe- prove production efficiencies of recombinant proteins, it is sized cathepsin B was translated in a pre-pro form and fi- necessary to investigate the quality control system of the nally activated in the lysosome (Hanewinkel et al., 1987). silk gland. This study demonstrates that the expression of This suggests that these inactive forms are used to supply mutant L chain, which does not contribute to producing lysosomal cathepsin B for degradation of misfolded fibro- normal fibroin elementary units, induces expression of ins in the lysosome. chaperones, such as BiP and Hsp20.8, in PSGs of the There are two types of stress-induced degradation of Nd-sD strain. Therefore, we expect that the PSG of the unfolded proteins in the lysosome: macroautophagy and Nd-sD strain will be a valuable tool for examining the chaperone-mediated autophagy (CMA) (Majeski and Dice, mechanisms of protein quality control in silkworms. 2004). Macroautophagy degrades mutant proteins in the lysosome, whereas the degradation pathway using CMA ACKNOWLEDGMENTS occurs in a substrate-specific manner. 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