The incubation of HeLa cells at 15°C induces the formation of Golgi tubules, which contain glycosylation enzymes but neither cargo nor matrix proteins. We now show by immunofluorescence and immunoelectron microscopy that these tubules are enriched in a specific set of SNARE and Rab proteins mediating intra‐Golgi transport (Gos28, GS15 and Rab6) but excluded others involved in endoplasmic reticulum–Golgi trafficking (Sec22, membrin, Rab 1 and Rab2). In vivo experiments using cyan fluorescent protein‐tagged galactosyltransferase showed that most of these tubules are dynamic transient membranes that grow to the cell periphery but then decrease until disappearing into the perinuclear area. Interestingly, in experiments carried out with cells cultured under physiological conditions, Golgi tubules containing Gos28, GS15, Rab6 and glycosylation enzymes and showing in vivo dynamics identical to that detected in low‐temperature‐cultured cells were observed. Together, our results support that low‐temperature‐induced tubules may be representatives of the carriers mediating intra‐Golgi recycling of enzymes.
OGP, encoded by the Ovgp1 gene, is the major non-serum oviductal protein in most mammals. In the genome of Rattus norvegicus, Ovgp1 has been identified as a pseudogene. However, Mus musculus presents a functional gene. As the rat and the mouse belong to the subfamily Murinae, Ovgp1 has probably been lost after their divergence. This study aims to determine when the pseudogenization event occurred and which proteins could replace its function. To attain that, the potential expression of members belonging to the GH18 family is investigated in the rat oviduct by means of molecular and proteomic analyses. Specific Ovgp1 regions are sequenced in different murine rodent species. The analysis reveals the presence of stop codons only in some species of the Rattini tribe, suggesting that the majority of the murine species present a functional gene. Thus, the pseudogenization of Ovgp1 could be dated back to around 10 Mya, after the divergence of the Rattini tribe. The expression of several genes and proteins of the GH18 family, such as Chia, Chit1, Chi3l1, and Chid1, are detected in the rat oviduct. This study opens the door for further research on GH18 family proteins that mimic the OGP functions in species where Ovgp1 is pseudogenized.
26 September 2000 in Molecular Reproduction and Development