Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles

Abstract

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their 9-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: First, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies. Second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during metazoan evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.

GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation

“…However, analysis of a pathogenic GO mutation that disrupts Golgi targeting (A220P) showed no effect upon GORAB function at the centriole. This finding is consistent with Golgi dysfunction being the primary cause of GO, although we cannot exclude an involvement of centriolar defects in GO pathology, possibly through defects at the cilium 30,31 . Interestingly, interference with Golgi targeting of Drosophila Gorab resulted in a spermatogenesis defect very similar to that seen in COPI-deficient flies, consistent with a functional association between GORAB and COPI being conserved in evolution 31,68 .…”

“…A recent study using both Drosophila embryos and human tissue culture cells has uncovered a role for GORAB in centriole duplication, which is distinct from its function at the Golgi apparatus 31 . This suggests that centriolar defects may contribute to the GO phenotype.…”

“…It has been proposed that GORAB is a member of the golgin family of coiled-coil Golgi proteins 22 , which participate in vesicle tethering 26,27 . GORAB has also been proposed to function as a transcriptional activator for neurite outgrowth 28 , as a modulator of MDM2 ubiquitylation that in turn can impact upon p53 levels and apoptosis 29 , and has recently been shown to play a role in centriole duplication and ciliogenesis 30,31 . Despite these advances, the function of Golgi-associated GORAB remains poorly defined, and the pathogenic mechanism underlying GO remains to be determined.…”

Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis

“…Gateway system (Thermo Fisher Scientific)-compatible Rcd4 entry clones were generated by amplifying the coding sequence from SD16838 cDNA clone (Drosophila Genomics Resource Center) and cloning into a pDONR221 entry vector (Invitrogen). The entry clones were recombined into pPGWattB, pUGWattB, pUWGattB Drosophila destination vectors (Kovacs et al, 2018). Transgenic ana3 fly lines for localization studies were similarly generated by integrating the transgene into the attP2 landing site (see Ana3 CDS synthesis).…”

The Close Relationship between the Golgi Trafficking Machinery and Protein Glycosylation

“…Evidence indicates that GORAB is a key regulator of the E3 ligase Mdm2, promoting both its transcription, by binding Mdm2 promoter [ 89 ], and Mdm2 protein self-ubiquitylation [ 90 ]. A recent work implicated the Drosophila homologue of GORAB in centriole structure and duplication [ 91 ]. Although the above studies proved a subcellular localization of GORAB in the cytoplasm and in the nucleus, the GO disease has been mostly associated with Golgi-associated functions [ 87 , 92 ].…”