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Tail-specific antibodies that block return of 46,000 M(r) mannose 6-phosphate receptor to the trans-Golgi network
C Schulze-Garg1, C Böker, S K Nadimpalli
1Universität Göttingen, Federal Republic of Germany.
Abstract:
Recycling of 46,000 M(r) 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) efficiently 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. F., 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.
Insights
Recycling of mannose 6-phosphate receptor (MPR 46) was blocked by targeting a specific peptide epitope. This led to MPR 46 accumulation in a specialized endosome, separate from lysosomal pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Trafficking
Background:
- The mannose 6-phosphate receptor (MPR 46) is crucial for lysosomal enzyme targeting.
- Understanding MPR 46 recycling is key to cellular protein sorting.
- The cytoplasmic domain of MPR 46 contains signals regulating its trafficking.
Purpose of the Study:
- To investigate the recycling pathway of MPR 46.
- To identify specific regions within the MPR 46 cytoplasmic domain involved in its endosomal sorting.
- To elucidate the mechanism by which MPR 46 is segregated from endocytosed cargo.
Main Methods:
- Microinjection of Fab fragments targeting specific MPR 46 cytoplasmic epitopes.
- Immunofluorescence microscopy to track receptor localization.
- Colocalization studies with endosomal markers and fluid-phase markers.
Main Results:
- Fab fragments against peptide 43-47 (Ala-Tyr-Arg-Gly-Val) blocked MPR 46 return to the TGN.
- MPR 46 accumulated in an endosomal compartment distinct from early (Rab5) and late (Rab7) endosomes.
- Accumulated MPR 46 did not colocalize with fluid-phase markers, indicating segregation from lysosomal pathways.
- Tyrosine residue 44 within peptide 43-47 is implicated as part of an internalization and/or sorting signal.
Conclusions:
- A specific epitope (peptide 43-47) in the MPR 46 cytoplasmic tail regulates its recycling.
- MPR 46 utilizes a specialized endosomal subcompartment for sorting.
- Tyrosine 44 is likely a critical component of a multifunctional sorting signal for MPR 46 trafficking.