br STAR Methods br Acknowledgments We would like to thank
Acknowledgments We would like to thank Dr. Tsung-Ping and Dr. Shang-Yi Tsai, National Institute on Drug Abuse, NIH for sharing protocols on pulse chase experiments and analysis. We would like to thank Dr. William G. Telford for his valuable input on Amnis flow cytometry experiments and analysis. We would like to thank Dr. Billur Akkaya, NIAID, for providing us with B6. Tbx21ZsGreenFoxp3RFP mice for chronic LCMV experiments. We would like to thank Dr. Bishop Hague, NIAID, for his assistance in flow cytometry sorting of virus-infected cells. We would like to thank the core facilities at Newcastle University namely Bioimaging, Comparative Biology Centre, and Flow cytometry. We would also like to thank Mr. Christopher Huggins for his technical expertise. The authors would also like to thank the NIH tetramer core facility for providing the tetramers used in this study. This research was funded by the Division of intramural research program of the NCI and NIAID, NIH, USA, Newcastle University Research Fellowship, Newcastle University, MRC-Newcastle University Single Cell Unit Award and the Academy of Medical Sciences, Springboard Award SBF003\\1129; G.M. is supported by MRC-DiMEN Doctoral Training Partnership program; J.M.-F. and C.W. are supported by the Wellcome Trust, S.I.v.K. is supported by an ERC starter grant ER-StG-639005, and A.L. is supported by Crohn\'s and Colitis Foundation of America.
Introduction Seed storage proteins are a source of trovafloxacin that are indispensable for seedling development during germination. They are stored in specialised tissues during seed development (Shutov et al., 2003). In cereals, the tissue that stores proteins is the endosperm. Cysteine proteinases from the papain family (C1A) are the main group of enzymes that degrade the storage proteins of cereal caryopses during germination (Prabucka and Bielawski, 2004), as confirmed by the rapid increase in their activity observed during the germination process (Bielawski et al., 1994). The active site of papain endopeptidases consists of three highly conserved amino acids: Cys 25 (numbers refer to the papain numbering), His 159 and Asn 175 (Wiederanders et al., 2003), although Beers et al. (2000) mention only Cys and His as the amino acids forming the catalytic dyad and Asn or Asp and His as two other residues important for catalysis. A special feature of papain endopeptidases is their a priori readiness for catalysis, resulting from a constant ionisation, independent of the substrate\'s presence, of cysteine, which is the nucleophile during peptide bond hydrolysis (Wiederanders et al., 2003). Cysteine proteases are synthesised as inactive or less active precursors, composed of an N-terminal signal sequence followed by a prosequence and a mature enzyme sequence. The tertiary structure of cysteine proteinases (CPs) consists of two domains (R and L) that are comparable in size, separated by the active site, which is previously “formed” in the precursor. The above-mentioned N-terminal prodomain, which exists irrespective of the initial sequence, contains an inhibitory sequence (I29 - MEROPS database) that extends along the substrate binding site and occupies positions S and S′ of the enzyme (Beers et al., 2000), thereby masking the enzyme\'s active site. Apart from its inhibitory function, the N-terminal prosequence is also thought to be responsible for the correct folding and stabilisation of the conformation in neutral and alkaline environments (Beers et al., 2000) as well as for targeting enzymes that contain the NIPR sequence to vacuoles (Kiyosaki et al., 2009), while the C-terminal K/H/TDEL sequence functions as the ER retention signal, which was also described by Okamoto et al. (2003) as participating in the formation of ER vesicles and involved in the efficient vacuolar transport of sulfhydryl-endopeptidase. Furthermore, a characteristic feature of plant papain-type endopeptidases, similar to human H and L cathepsins, is the presence of the discontinuous ERFNIN motif in the N-terminal prosequence, which distinguishes them from plant B-like cathepsins (Karrer et al., 1993). Papain-like CPs were subclassified in detail and characterised by Richau et al. (2012).