We have studied the intracellular trafficking of the envelope glycoprotein I (gpI) of the varicella‐zoster virus, a human herpes virus whose assembly is believed to occur in the trans‐Golgi network (TGN) and/or in endocytic compartments. When expressed in HeLa cells in the absence of additional virally encoded factors, this type‐I membrane protein localizes to the TGN and cycles between this compartment and the cell surface. The expression of gpI promotes the recruitment of the AP‐1 Golgi‐specific assembly proteins onto TGN membranes, strongly suggesting that gpI, like the mannose 6‐phosphate receptors, can leave the TGN in clathrin‐coated vesicles for subsequent transport to endosomes. Its return from the cell surface to the TGN also occurs through endosomes. The transfer of the gpI cytoplasmic domain onto a reporter molecule shows that this domain is sufficient to confer TGN localization. Mutational analysis of this domain indicates that proper subcellular localization and cycling of gpI depend on two different determinants, a tyrosine‐containing tetrapeptide related to endocytosis sorting signals and a cluster of acidic amino acids containing casein kinase II phosphorylatable residues. Thus, the VZV gpI and the mannose 6‐phosphate receptors, albeit localized in different intracellular compartments at steady‐state, follow similar trafficking pathways and share similar sorting mechanisms.

Compartmentalized bacteria have proteins that are structurally related to eukaryotic membrane coats, and one of these proteins localizes at the membrane of vesicles formed inside bacterial cells.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6‐phosphate signal by two receptors: the cation‐independent mannose 6‐phosphate/insulin‐like growth factor II receptor (CI‐MPR) and the cation‐dependent mannose 6‐phosphate receptor (CD‐MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD‐MPR‐deficient mice with Thp mice that carry a CI‐MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI‐MPR, like those lacking the CD‐MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6‐phosphate binding proteins in mammalian cells.

In mammalian cells, the mannose 6-phosphate receptors (MPRs) and the lysosomal glycoproteins, lysosomalassociated membrane protein (LAMP) I, lysosomal integral membrane protein (LIMP) II, are directly transported from the trans-Golgi network to endosomes and lysosomes. While MPR traffic relies on the AP-1 adaptor complex, we report that proper targeting of LAMP I and LIMP II to lysosomes requires the AP-3 adaptor-like complex. Overexpression of these proteins, which contain either a tyrosine-or a di-leucine-basedsorting motif, promotes AP-3 recruitment on membranes. Inhibition of AP-3 function using antisense oligonucleotides leads to a selective misrouting of both LAMP I and LIMP II to the cell surface without affecting MPR trafficking. These results provide evidence that AP-3 functions in the intracellular targeting of transmembrane glycoproteins to lysosomes.