Rabaptin4, a novel effector of the small GTPase rab4a, is recruited to perinuclear recycling vesicles.

The small GTPase rab4a is associated with early endocytic compartments and regulates receptor recycling from early endosomes. To understand how rab4a mediates its function, we searched for proteins which associate with this GTPase and regulate its activity in endocytic transport. Here we identified rabaptin4, a novel effector molecule of rab4a. Rabaptin4 is homologous with rabaptin5 and contains a C-terminal deletion with respect to rabaptin5. Rabaptin4 preferentially interacts with rab4a-GTP and to a lesser extent with rab5aGTP. We identified a rab4a-binding domain in the N-terminal region of rabaptin4, and two binding sites for rab5, including a novel N-terminal rab5a-binding site. Rabaptin4 is a cytosolic protein that inhibits the intrinsic GTP hydrolysis rate of rab4a and is recruited by rab4a-GTP to recycling endosomes enriched in cellubrevin and internalized indocarbocyanine-3 (Cy3)-labelled transferrin. We propose that rabaptin4 assists in the docking of transport vesicles en route from early endosomes to recycling endosomes.

Folding of viral envelope glycoproteins in the endoplasmic reticulum.

Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality control system of the endoplasmic reticulum. A special characteristic that distinguishes viral fusion proteins from most cellular proteins is the extensive conformational change they undergo during fusion of the viral and cellular membrane. Many viral proteins fold in conjunction with and dependent on a viral partner protein, sometimes even synthesized from the same mRNA. Relevant for folding is that viral glycoproteins from the same or related virus families may consist of overlapping sets of domain modules. The consequences of these features for viral protein folding are at the heart of this review.

Structure-function analysis of the gE-gI complex of feline herpesvirus: mapping of gI domains required for gE-gI interaction, intracellular transport, and cell-to-cell spread.

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.

Biosynthesis of glycoproteins E and I of feline herpesvirus: gE-gI interaction is required for intracellular transport.

The biosynthesis of glycoproteins E and I of feline herpesvirus was studied by using the vaccinia virus vTF7-3 expression system. gE and gI were synthesized as N-glycosylated, endoglycosidase H (EndoH)-sensitive precursors with Mrs of 83,000 and 67,000, respectively. When coexpressed, gE and gI formed sodium dodecyl sulfate-sensitive hetero-oligomeric complexes that were readily transported from the endoplasmic reticulum (ER). Concomitantly, the glycoproteins acquired extensive posttranslational modifications, including O glycosylation, leading to an increase in their apparent molecular weights to 95,000 and 80,000 to 100,000 for gE and gI, respectively. In the absence of gE, most gI remained EndoH sensitive. Only a minor population became EndoH resistant, but these molecules were processed aberrantly as indicated by their Mrs (100,000 to 120,000). By immunofluorescence microscopy, gI was detected primarily in the ER but also at the plasma membrane. gE, when expressed by itself, remained EndoH sensitive and was found only in the ER and the nuclear envelope. These results were corroborated by studying the biosynthesis of gE in feline herpesvirus (FHV)-infected cells. In cells infected with wild-type FHV, gE acquired the same co- and posttranslational modifications as during vTF7-3-driven expression. However, an FHV mutant lacking gI failed to produce mature gE. We conclude that gE is retained in the ER, presumably by associating with molecular chaperones, and becomes transport competent only when in a complex with gI.