Tail-specific Antibodies That Block Return of 46,000 Mr Mannose 6-Phosphate Receptor to the trans-Golgi Network

Recycling of 46,000 Mr mannose 6-phosphate receptor (MPR 46) was investigated by microinjection of Fab fragments against small epitopes within the cytoplasmic domain of the receptor. Fab fragments against the peptide 43-47 (Ala-Tyr-Arg-Gly-Val) efticiently blocked return of MPR 46 to the TGN. Antibody-induced redistribution resulted in accumulation of MPR 46 within an endosomal compartment, from which it recycled to the plasma membrane. Rab5 and rab7, markers for early and late endosomes, respectively, were not detectable in the compartment of redistributed MPR 46, suggesting that it represents a specialized endosomal subcompartment. The bulk of redistributed MPR 46 did not colocalize with endocytosed fluid-phase marker, suggesting that it accumulates at a site where MPR 46 has been segregated from endocytosed material, which is destined for transport to lysosomes. Peptide 43-47 contains a tyrosine (residue 44) which has been shown earlier to be part of an internalization signal for MPR 46 (Johnson, K. E, W. Chan, and S. Kornfeld. 1990. Proc. Natl. Acad. Sci. USA. 87:10010-10014). The role of tyrosine residue 44 as part of a putative multifunctional sorting signal is discussed. M PR 46 (46,000 Mr mannose 6-phosphate receptor) ~ is one of the two receptors that are involved in sorting and transport of newly synthesized lysosomal enzymes (for review see Kornfeld and MeUman, 1989). 46,000 Mr MPR (MPR 46) is thought to bind lysosomal enzymes in the TGN for segregation from resident Golgi proteins and from proteins of the secretory pathway. After transfer to the endosomal compartment, receptor-ligand complexes dissociate due to the acidic pH. Lysosomal enzymes reach their final destination by an unknown mechanism, whereas MPR 46 recycles back to the TGN. While sorting of newly synthesized lysosomal enzymes towards lysosomes is carried out by both, MPR 46 and the 300,000 Mr MPR (MPR 300), only MPR 46 mediates export of a portion of the lysosomal enzymes (Chao et al., 1990). On the other hand, binding of lysosomal enzymes at the cell surface and receptor-mediated endocytosis is a unique function of MPR 300 (Stein et al., 1987). Nevertheless, MPR 46 is transported to the plasma membrane and recycles between plasma membrane and endosomes. The functional diversity of the two MPRs is reflected by their overlapping but not identical subcellular distribution. By immunoelectron microscopy, both receptors were found in the TGN and in early as well as late endosomes (Bleekemolen et al., 1988; Matovcik et al., 1990). In the enS. K. Nadimpalli's present address is School of Life Sciences, University of Hyderabad, Central University P.O., Hyderabad-500 134, India. 1. Abbreviation used in this paper: MPR, marmose-6 phosphate receptor. dosomal compartment, however, partial segregation of the two receptors has been observed (Klumperman et al., 1993). MPR 300 was found to be enriched in the large vacuolar part of endosomes, when compared with associated tubular extensions and small vesicles surrounding the endosomes (referred to as ATV). The ratio of immunogold-labeled MPR 46 over MPR 300 was higher in ATV than in the vacuolar part of the endosomes, suggesting differential sorting of the two receptors. In most instances, sorting of receptors is regulated by signals in their cytoplasmic domain, which are recognized by cytosolic transport factors. Though recently it has been postulated that also the extraceUular or transmembrane domains play a role for recycling of MPR 300, the signals involved have not been identified to date (Dintzis and Pfeffer, 1990). The sorting of receptors into clathrin-coated pits of the plasma membrane depends on a tyrosine residue, or an aromatic amino acid as part of an internalization signal in their cytoplasmic domain. This signal is recognized by HA-2 adaptors, the protein complex which induces the assembly of the clathrin coat around receptors at the plasma membrane (for review see Pearse and Robinson, 1990). The tyrosine-containing internalization signal is thought to be exposed in a B-turn conformation within the cytoplasmic domain as shown for low density lipoprotein (LDL) receptor, transferrin receptor, and lysosomal acid phosphatase (Bansal and Gierasch, 1991; Collawn et al., 1990; Ktistakis et al., 1990; Eberle et al., 1991). For the rapid internalization of MPR 46, a more complex signal has been characterized by © The Rockefeller University Press, 0021-9525/93/08/541/11 $2.00 The Journal of Cell Biology, Volume 122, Number 3, August 1993 541-551 541 on A uust 3, 2017 jcb.rress.org D ow nladed fom site-directed mutagenesis. Johnson et al. (1990) suggested two distinct internalization signals in the cytoplasmic domain of bovine MPR 46, the first signal including amino acids Phe-12 and Phe-17, and the second including Tyr-44. For the rapid endocytosis of human MPR 46 the last seven amino acids of the cytoplasmic domain proved to be essential (Weber et al., in preparation). A second clathrin-dependent pathway is involved in transport from the TGN to the endosomal compartment. HA1 adaptors, which are thought to mediate assembly of coated vesicles at the TGN, have been shown to bind to an as yet unidentified signal in the cytoplasmic domain of MPR 300 and MPR 46 (Glickman et al., 1989; Sosa et al., 1993). Additional signals that regulate exit from the endosomal compartment and recycling of receptors to the TGN or to the plasma membrane, and putative cytosolic proteins involved in these sorting steps remain to be characterized. We have used peptide-specific antibodies that recognize small peptide epitopes within the cytoplasmic domain of human MPR 46 to screen for sorting signals involved in its intraceUular transport. In the present study we show that F fragments against peptide 38-52 of the cytoplasmic domain of MPR 46, when microinjected into human fibroblasts, prevent recycling of MPR 46 to the TGN, resulting in accumulation of MPR 46 in a vesicular compartment that is related to endosomes. Antibodies against the peptide Ala-Tyr-ArgGly-Val, which constitutes the central epitope of peptide 38-52, were most efficient in causing this redistribution. Antibodies against peptide 7-21 did not alter the distribution of MPR 46. Materials and Methods