Resultados de la variable única

Soluble proteins destined for the degradative organelle, the lysosome, are selectively concentrated into budding regions of TGN termed "coated pits". Vesicles bud off into the cytoplasm and fuse with endocytic compartments, which finally deliver lysosomal

enzymes to lysosomes. In addition, it is thought that a proportion of these proteins may pass to the cell surface before entering lysosomes (extensively reviewed by Komfeld and Mellman, 1989; Pearse and Robinson, 1990; Trowbridge etal., 1993).

The targeting of soluble proteins to the yeast lysosome equivalent, the vacuole, involves the recognition of a propeptide sequence at the protein N-terminus. Although a QPRL sequence has been identified as critical for the vacuolar localisation of carboxypeptidase Y (Vails et al., 1990), no significant homology can be found between the propeptides of vacuolar enzymes and it may be that a common teitiary structure or signal patch is the sorting determinant in this case (Komfeld, 1987).

In mammalian cells sorting of soluble lysosomal proteins (such as cathepsin D) into coated vesicles for intracellular delivery to lysosomes involves an extensively characterised receptor mediated mechanism. The protein coat associated with these vesicles and with the TGN coated pits consists of an outer lattice of clathrin and an inner shell of adaptor protein complexes (figure 1.3.). It is these adaptors that interact with the cytoplasmic domains of the receptors in the membrane, which in tum interact with their

l i g h t .c h a i n h e a v y c h a i n d i s t a l 50K

Plasm a membrane adaptor HA-2

47K

A d a p tin A d a p tin

Golgi adaptor HA-1

Figure 1.3. The structure and protein composition o f the clathrin coat {Pearseet al.,

1990).

Upper left: Schematic drawing showing the modular structure of the triskelion.

Upper right: Diagram showing how triskelions form a hexagonal lattice.

Lower: Diagrammatic representation of the HA-2/AP-2 adaptor in

ligands in the vesicle lumen and mediate segregation of lysosomal enzymes (Keen, 1990; Pearse and Robinson, 1990; Robinson, 1992; Takizawa and Malhotra, 1993).

The mannose 6-phosphate groups added to lysosomal hydrolases in the cis Golgi (section 1.1.3.) now serve as a recognition marker for a mannose 6-phosphate receptor in the TGN. Two of these receptors exist: One of 215kD, which binds M6P independently of divalent cations (CI-MPR); and one of 46kD, which shows enhanced M6P binding in the presence of divalent cations (CD-MPR). Both these receptors bind the M6P moiety on soluble lysosomal proteins within the mildly acidic lumen of the TGN, with the CI-MPR being the dominant receptor in this process (for discussion see Komfeld, 1992; Komfeld and Mellman, 1989). CI-MPR has been shown to recmit the TGN-specific adaptor protein HA-l/AP-1 onto TGN membranes when bound to M6P residues (LeBorgne et al.y 1993; Méresse et al., 1990), which in tum promotes the recmitment of the clathrin onto TGN membranes (Keen, 1990; Pearse and Robinson, 1990; Robinson, 1992). A coated vesicle then buds away from the TGN, thereby segregating soluble lysosomal enzymes from proteins destined for secretion (Klumperman et at., 1993; Lemansky etal.,

1987; Schulze-Lohoff et al., 1995). When these coated vesicles are delivered to an acidified endosomal compartment (the sorting endosome and/or prelysosome; sections 1.2.3. and 1.2.4.), the low pH induces the dissociation of lysosomal hydrolases from the MPRs. The hydrolases then pass to the lysosome, where the phosphate group may be removed (Ludwig et a l.,\9 9 \) and the enzymes become fully functional at the acidic pH of 4.5-5.0 (Komfeld and Mellman, 1989; Mellman et al., 1986). MPRs do not enter the lysosome but recycle back to the TGN and undergo many rounds of delivery (Komfeld, 1987; Pfeffer, 1987).

A minor proportion of the CI-MPR has also been found to recycle from the TGN via the cell surface to endocytic compartments (Stoorvogel et a l, 1989). At the cell surface the CI-MPR may bind the small proportion of lysosomal enzymes that are constitutively secreted from the cell (Lemansky et al., 1987), but it can also act as a receptor for insulin­ like growth factor II (Komfeld, 1992; Komfeld and Mellman, 1989). Cell surface CI-

MPR is incorporated into clathrin coated pits that contain the cell surface-specific adaptor protein HA-2/AP-2 within their cytoplasmic protein coat The CI-MPR cytoplasmic tail thus contains two targeting signals, one that interacts with HA-1 adaptors at the TGN (this is reported to involve the transient phosphorylation of serine residues) (LeBorgne et al.j

1993) and one that interacts with HA-2 adaptors at the plasma membrane (for review see Komfeld, 1987). These signals are thought to behave independently, with a di-leucine motif being critical for the intracellular transport of lysosomal enzymes and a YXKV sequence pattern acting as an internalisation signal for endocytosis (Chen et. a l, 1993; Johnson and Komfeld, 1992b; Trowbridge e ta l, 1993 ; section 1.2.1.). An HLL motif in the cytoplasmic tail of the CD-MPR is also essential for the intracellular routing of lysosomal hydrolases (Johnson and Komfeld, 1992a).

In contrast to the mechanism described above, the enzyme lysosomal acid phosphatase (LAP) is transported to the cell surface via the constitutive pathway as an integral type 1 membrane protein. It then recycles in and out of endocytic compartments at least twelve times before finally arriving in lysosomes, where it is processed to form a soluble lysosomal enzyme (Braun e ta l, 1989; Peters et al., 1990).