Sorting of lysosomal membrane glycoproteins lamp-1 and lamp-2 into vesicles distinct from mannose 6-phosphate receptor/gamma-adaptin vesicles at the trans-Golgi network.
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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 6-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. (+info)
Identification and characterization of novel clathrin adaptor-related proteins.
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We have identified a human approximately 87-kDa protein, designated as gamma2-adaptin, that is similar to gamma-adaptin (called gamma1-adaptin in this paper), a large chain of the AP-1 clathrin-associated adaptor complex, not only in the primary structure (60% amino acid identity) but also in the domain organization. Northern blot analysis has shown that its mRNA is expressed in a variety of tissues. Analysis using a yeast two-hybrid system has revealed that, similarly to gamma1-adaptin, gamma2-adaptin is capable of interacting not only with the sigma1 chain (called as sigma1A in this paper), the small chain of the AP-1 complex, but also with a novel sigma1-like protein, designated as sigma1B, which shows an 87% amino acid identity to sigma1A; and that, unlike gamma1-adaptin, it is unable to interact with beta1-adaptin, another large chain of the AP-1 complex. Immunofluorescence microscopy analysis has revealed that gamma2-adaptin is localized to paranuclear vesicular structures that are not superimposed on structures containing gamma1-adaptin. Furthermore, unlike gamma1-adaptin, gamma2-adaptin is recruited onto membranes in the presence of a fungal antibiotic, brefeldin A. These data suggest that gamma2-adaptin constitute a novel adaptor-related complex that participates in a transport step different from that of AP-1. (+info)
Cloning, expression, and localization of a novel gamma-adaptin-like molecule.
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We describe the cloning, expression, and localization of gamma2-adaptin, a novel isoform of gamma-adaptin. The predicted human and mouse gamma2-adaptin proteins are approximately 90 kDa and 64.4% and 61.7%) identical to gamma-adaptin, respectively. gamma2-Adaptin was localized to the Golgi, its localization distinct from gamma-adaptin. The membrane association of gamma- and gamma2-adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, gamma2-adaptin binding being insensitive to drug treatment. Together, these results suggest that gamma2-adaptin plays a role in membrane transport distinct from that played by gamma-adaptin. (+info)
Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.
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Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN. (+info)
ADP-ribosylation factor-1 is sensitive to N-ethylmaleimide.
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The treatment of normal rat kidney cells with N-ethylmaleimide caused the release of beta-COP, a component of coatomer, from the Golgi apparatus without causing disassembly of the organelle. The release of beta-COP, which was not due to depolymerization of microtubules, was markedly blocked by the activation of GTP-binding proteins by aluminum fluoride or a nonhydrolyzable analogue of GTP. To determine which component is N-ethylmaleimide-sensitive, we reconstituted the recruitment of coatomer from the bovine brain cytosol onto the Golgi apparatus in digitonin-permeabilized cells. In cells treated with N-ethylmaleimide before permeabilization, beta-COP was still recruited onto the Golgi apparatus. In contrast, beta-COP was not recruited when N-ethylmaleimide-treated bovine brain cytosol was used. These results suggest that the N-ethylmaleimide-sensitive factor(s) are present in the cytosol. It is known that coatomer and ADP-ribosylation factor-1 (ARF1) are the only cytoplasmic proteins needed for the assembly of Golgi-derived coated vesicles. N-Ethylmaleimide treatment of a coatomer-rich fraction did not affect the binding of beta-COP to the Golgi apparatus, whereas the same treatment of an ARF-rich fraction abolished beta-COP binding. Similar results were obtained using purified recombinant ARF1. Concomitant with inactivation, 0.85 mol of N-ethylmaleimide was incorporated into 1 mol of ARF1. ARF1 contains only one cysteine residue (Cys-159), which is located near the base moiety of the bound guanine nucleotide. (+info)