Sorting of lysosomal membrane glycoproteins lamp-1 and lamp-2 into vesicles distinct from mannose 6-phosphate receptor/γ-adaptin vesicles at the trans-Golgi network

Newly synthesized lysosomal membrane glycoproteins lamp-1 and lamp-2 are primarily sorted at the trans-Golgi network (TGN) by recognition of a tyrosine-based signal sequence in their cytoplasmic tails. It is presently unclear how this signal is recognized and what type of vesicle transports lamp-1 and lamp-2. Here, we describe a method to generate transport vesicles containing lamp proteins from the TGN in vitro. The method is based on incorporation of radioactive sialic acid in glycoproteins at the TGN by incubation of membranes with tritiated CMP-sialic acid. The generation of vesicles from labeled membranes required ATP and cytosol, and was temperature-dependent and brefeldin A-sensitive. Analysis on Nycodenz gradients revealed that lamp-vesicles were distinct from vesicles containing gamma-adaptin and mannose B-phosphate receptor (MPR), Moreover, both these types of vesicles migrated differently than vesicles containing proteins destined for the plasma membrane. The conclusion that lamps and MPRs are sorted into different vesicles was further strengthened by the finding that whereas wortmannin both in vitro and in vivo inhibited the production of gamma-adaptin/MPR-containing vesicles, this drug had no effect on the generation of lamp-vesicles and on the sorting of lamps. The results indicate that membrane proteins containing tyrosine-based motifs for sorting at the TGN are segregated from clathrin-coated vesicles containing MPRs.