Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous ␥-secretase cleavage of the ␤-amyloid precursor protein and signaling receptor Notch. PS holoprotein undergoes endoproteolysis by an unknown enzymatic activity to generate NH 2 -and COOH-terminal fragments, a process that is required for the formation of the active and stable PS/-␥-secretase complex. Biochemical and genetic studies have recently identified nicastrin, APH-1, and PEN-2 as essential cofactors that physically interact with PS1 and are necessary for the ␥-secretase activity. However, their precise function in regulating the PS complex and ␥-secretase activity remains unknown. Here, we demonstrate that endogenous PEN-2 preferentially interacts with PS1 holoprotein. Down-regulation of PEN-2 expression by small interfering RNA (siRNA) abolishes the endoproteolysis of PS1, whereas overexpression of PEN-2 promotes the production of PS1 fragments, indicating a critical role for PEN-2 in PS1 endoproteolysis. Interestingly, accumulation of full-length PS1 resulting from down-regulation of PEN-2 is alleviated by additional siRNA down-regulation of APH-1. Furthermore, overexpression of APH-1 facilitates PEN-2-mediated PS1 proteolysis, resulting in a significant increase in PS1 fragments. Our data reveal a direct role of PEN-2 in proteolytic cleavage of PS1 and a regulatory function of APH-1, in coordination with PEN-2, in the biogenesis of the PS1 complex.Alzheimer's disease is a progressive neurodegenerative disorder that is pathologically characterized by the cerebral deposition of 39 -42 amino acid peptides, termed ␤-amyloid (A␤) 1 (1). A␤ peptides are proteolytically derived from amyloid precursor protein (APP), a type 1 transmembrane glycoprotein that predominantly resides in the trans-Golgi network (TGN), by two distinct enzymatic activities known as ␤-secretase (or BACE) and ␥-secretase (2). Mutations of presenilins (presenilin 1 (PS1) and presenilin 2 (PS2)) are responsible for the majority of early-onset familial Alzheimer's disease. Several lines of evidence suggest that PSs play a crucial role in intramembranous ␥-secretase cleavage of select type I membrane proteins including APP and the signaling receptor, Notch-1 (3-9).PS1 encodes a hydrophobic protein with 467 amino acids spanning the membrane six to eight times (10, 11). Shortly after being synthesized, PS1 undergoes endoproteolytic cleavage by unknown proteases (named presenilinases) between transmembrane domains 6 and 7 to generate a 27-kDa NH 2 -terminal fragment (NTF) and a 17-kDa COOH-terminal fragment (CTF) (12). Cell biological studies indicated that fulllength PS1 resides almost exclusively in the endoplasmic reticulum (ER), while the NTF and CTF localize predominantly in the Golgi and, to a lesser extent, in the plasma membrane and endocytic compartments (13-15). Endoproteolytic cleavage of PS1 is suggested to take place in the ER and the cleaved fragments are subsequently transported to the Golgi compartments (16). The subcellular location of the ␥-...

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.

␥-Secretase, which is responsible for the intramembranous cleavage of Alzheimer ␤-amyloid precursor protein and the signaling receptor Notch, is a multiprotein complex consisting of at least four components: presenilin (PS); nicastrin (Nct); APH-1 (anterior pharynx-defective-1); and presenilin enhancer-2 (PEN-2). Presenilin 1 (PS1) is known to be essential for the stability, interaction, and trafficking of the other PS1/␥-secretase components. However, the precise functions of the other components remain elusive. Here, we investigated the functions of Nct within the PS1/␥-secretase complex. We demonstrated that the loss of Nct expression in the embryonic fibroblast cells (Nct KO cells) results in dramatically decreased levels of APH-1, PEN-2, and PS1 fragments accompanied by a significant accumulation of full-length PS1. In the absence of Nct, PEN-2 and fulllength PS1 are subjected to proteasome-mediated degradation, whereas the degradation of APH-1 is mediated by both proteasomal and lysosomal pathways. Unlike the case of wild type cells in which the ␥-secretase complex mainly locates in the trans-Golgi network, the majority of residual PEN-2, APH-1, and the uncleaved fulllength PS1 in Nct KO cells reside in the endoplasmic reticulum, which remain associated with each other in the absence of Nct. Interestingly, significant amounts of full-length PS1 and PEN-2, but not APH-1, are detected on the plasma membrane in Nct KO cells, suggesting the Nct-independent cell surface delivery of the PEN-2⅐PS1. Finally, the diminished PEN-2 protein level in Nct-deficient cells can be partially restored by overexpression of exogenous PS1, APH-1, or PEN-2 individually or collectively, indicating a dispensable role for Nct in controlling PEN-2 level. Taken together, our study demonstrates a critical role of Nct in the stability and proper intracellular trafficking of other components of the PS1/ ␥-secretase complex but not in maintaining the association of PEN-2, APH-1, and full-length PS1.

The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37°C. Several vps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, and vps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36 cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 and vps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (and vps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface in vps1 cells. We hypothesize that in vps8 and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.

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