In the secretory pathway, altering their molecular weight. Thus, progressive protease maturation is a marker that can pinpoint protease location during vacuolar transit.40,82 (The reader is referred to figure 2 in ref. 40 for a cartoon outlining the maturation events associated with the vacuolar proteases.) Perhaps the best-characterized example of a protease that follows these events is CPY. The CPY signal sequence targets the nascent protein to the ER and is then removed. ER lumenal CPY has a propeptide, which blocks protease activity and aids in CPY folding and glycosylation to generate p1-CPY.93,94 Further glycosylation in the Golgi forms the higher molecular weight p2-CPY species. Finally, in the vacuole Pep4 cleaves the CPY propeptide to produce the lower molecular weight, mature CPY enzyme.95 Defects in CPY processing were instrumental in identifying proteins involved in protein quality control, glycosylation, and trafficking pathways between the ER and vacuole. When Golgito-vacuolar trafficking is impaired, CPY is secreted into the periplasmic space, and several genetic screens cleverly exploited this feature to identify proteins required for this traffickinginterval.20,22,23,96-98 For example, the CPY sorting receptor, Vps10, which is required for Golgi-to-MVB trafficking, was first identified in one such screen, and additional sorting receptors have since been characterized.99,100 Unlike CPY, which requires the Vps10 receptor for Golgito-MVB trafficking, carboxypeptidase S (CPS) is produced as a precursor (pCPS) that is a type-II membrane protein.40,101 Once pCPS reaches the vacuole Pep4 or Prb1 cleave the transmembrane anchor to generate mature CPS (mCPS), which is a soluble peptidase in the vacuolar lumen.Fosinopril sodium 101 Therefore, CPS maturation can also be tracked by monitoring changes in the protein’s molecular weight.101,102 Studies using CPS demonstrated that Rsp5-mediated ubiquitination of cargos and recognition of the ubiquitinated species by the ESCRTs drives sorting to the vacuolar lumen.29,31,102,103 Efficient ubiquitination and segregation of CPS to the vacuolar lumen also requires adaptor proteins Tul1 and Bsd2, each of which contain L/PPxY motifs that recruit the Rsp5 ubiquitin ligase to CPS.104,105 This paradigm of ubiquitin ligase recruitment to substrates in the secretory pathway by adaptors is broadly conserved.34 In stark contrast to CPY and CPS, aminopeptidase 1 (referred to as Ape1 or API) does not enter the secretory pathway and lacks a signal sequence motif.Adalimumab 106 While CPY and CPS transit to the vacuole in 6 min, Ape1 requires over 40 min to mature.PMID:31085260 106,107 As noted above, Ape1 uses the Cvt pathway.52,106,108 The Cvt pathway requires similar machinery to the autophagy pathway and represents a de novo mechanism for vesicle formation in the cytoplasm.109,110 Soluble vacuolar proteases Proteinase A (PrA), encoded by the PEP4 gene, is a monomeric 42 kDa aspartyl endoprotease of the A1 family of proteases. PrA is targeted to the vacuole via the secretory pathway and is a key enzyme in the vacuolar protease activation cascade.1,40,111 Many vacuolar hydrolases, including PrA, are initially produced as inactive precursor forms, known as zymogens.112 Zymogens are not activated until they have been delivered to the vacuole, where an inhibitory propeptide is removed by proteolysis. This allows hydrolase activity to be sequestered within the vacuole, thereby protecting residents of the secretory pathway from proteolytic damage.