GOLPH3 protein controls organ growth by interacting with TOR signaling proteins in Drosophila.

The oncoprotein GOLPH3 (Golgi phosphoprotein 3) is an evolutionarily conserved phosphatidylinositol 4-phosphate effector, mainly localized to the Golgi apparatus, where it supports organelle architecture and vesicular trafficking. Overexpression of human GOLPH3 correlates with poor prognosis in several cancer types and is associated with enhanced signaling downstream of mTOR (mechanistic target of rapamycin). However, the molecular link between GOLPH3 and mTOR remains elusive. Studies in Drosophila melanogaster have shown that Translationally controlled tumor protein (Tctp) and 14-3-3 proteins are required for organ growth by supporting the function of the small GTPase Ras homolog enriched in the brain (Rheb) during mTORC1 (mTOR complex 1) signaling. Here we demonstrate that Drosophila GOLPH3 (dGOLPH3) physically interacts with Tctp and 14-3-3ζ. RNAi-mediated knockdown of dGOLPH3 reduces wing and eye size and enhances the phenotypes of Tctp RNAi. This phenotype is partially rescued by overexpression of Tctp, 14-3-3ζ, or Rheb. We also show that the Golgi localization of Rheb in Drosophila cells depends on dGOLPH3. Consistent with dGOLPH3 involvement in Rheb-mediated mTORC1 activation, depletion of dGOLPH3 also reduces levels of phosphorylated ribosomal S6 kinase, a downstream target of mTORC1. Finally, the autophagy flux and the expression of autophagic transcription factors of the TFEB family, which anti correlates with mTOR signaling, are compromised upon reduction of dGOLPH3. Overall, our data provide the first in vivo demonstration that GOLPH3 regulates organ growth by directly associating with mTOR signaling proteins.

Identification of GOLPH3 Partners in Drosophila Unveils Potential Novel Roles in Tumorigenesis and Neural Disorders.

Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. We recently showed that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. Here, we use affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, we found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. Our findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases.

The Close Relationship between the Golgi Trafficking Machinery and Protein Glycosylation.

Glycosylation is the most common post-translational modification of proteins; it mediates their correct folding and stability, as well as their transport through the secretory transport. Changes in N- and O-linked glycans have been associated with multiple pathological conditions including congenital disorders of glycosylation, inflammatory diseases and cancer. Glycoprotein glycosylation at the Golgi involves the coordinated action of hundreds of glycosyltransferases and glycosidases, which are maintained at the correct location through retrograde vesicle trafficking between Golgi cisternae. In this review, we describe the molecular machinery involved in vesicle trafficking and tethering at the Golgi apparatus and the effects of mutations in the context of glycan biosynthesis and human diseases.

A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila.

In animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. Here we demonstrate that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. We show that cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. Our results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. We propose that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.

Oncogenic Roles of GOLPH3 in the Physiopathology of Cancer.

Golgi phosphoprotein 3 (GOLPH3), a Phosphatidylinositol 4-Phosphate [PI(4)P] effector at the Golgi, is required for Golgi ribbon structure maintenance, vesicle trafficking and Golgi glycosylation. GOLPH3 has been validated as an oncoprotein through combining integrative genomics with clinopathological and functional analyses. It is frequently amplified in several solid tumor types including melanoma, lung cancer, breast cancer, glioma, and colorectal cancer. Overexpression of GOLPH3 correlates with poor prognosis in multiple tumor types including 52% of breast cancers and 41% to 53% of glioblastoma. Roles of GOLPH3 in tumorigenesis may correlate with several cellular activities including: (i) regulating Golgi-to-plasma membrane trafficking and contributing to malignant secretory phenotypes; (ii) controlling the internalization and recycling of key signaling molecules or increasing the glycosylation of cancer relevant glycoproteins; and (iii) influencing the DNA damage response and maintenance of genomic stability. Here we summarize current knowledge on the oncogenic pathways involving GOLPH3 in human cancer, GOLPH3 influence on tumor metabolism and surrounding stroma, and its possible role in tumor metastasis formation.

Drosophila Doublefault protein coordinates multiple events during male meiosis by controlling mRNA translation.

During the extended prophase of Drosophila gametogenesis, spermatocytes undergo robust gene transcription and store many transcripts in the cytoplasm in a repressed state, until translational activation of select mRNAs in later steps of spermatogenesis. Here, we characterize the Drosophila Doublefault (Dbf) protein as a C2H2 zinc-finger protein, primarily expressed in testes, that is required for normal meiotic division and spermiogenesis. Loss of Dbf causes premature centriole disengagement and affects spindle structure, chromosome segregation and cytokinesis. We show that Dbf interacts with the RNA-binding protein Syncrip/hnRNPQ, a key regulator of localized translation in Drosophila We propose that the pleiotropic effects of dbf loss-of-function mutants are associated with the requirement of dbf function for translation of specific transcripts in spermatocytes. In agreement with this hypothesis, Dbf protein binds cyclin B mRNA and is essential for translation of cyclin B in mature spermatocytes.

Modeling Congenital Disorders of N-Linked Glycoprotein Glycosylation in Drosophila melanogaster.

Protein glycosylation, the enzymatic addition of N-linked or O-linked glycans to proteins, serves crucial functions in animal cells and requires the action of glycosyltransferases, glycosidases and nucleotide-sugar transporters, localized in the endoplasmic reticulum and Golgi apparatus. Congenital Disorders of Glycosylation (CDGs) comprise a family of multisystemic diseases caused by mutations in genes encoding proteins involved in glycosylation pathways. CDGs are classified into two large groups. Type I CDGs affect the synthesis of the dolichol-linked Glc3Man9GlcNac2 precursor of N-linked glycosylation or its transfer to acceptor proteins. Type II CDG (CDG-II) diseases impair either the trimming of the N-linked oligosaccharide, the addition of terminal glycans or the biosynthesis of O-linked oligosaccharides, which occur in the Golgi apparatus. So far, over 100 distinct forms of CDGs are known, with the majority of them characterized by neurological defects including mental retardation, seizures and hypotonia. Yet, it is unclear how defective glycosylation causes the pathology of CDGs. This issue can be only addressed by developing animal models of specific CDGs. Drosophila melanogaster is emerging as a highly suitable organism for analyzing glycan-dependent functions in the central nervous system (CNS) and the involvement of N-glycosylation in neuropathologies. In this review we illustrate recent work that highlights the genetic and neurobiologic advantages offered by D. melanogaster for dissecting glycosylation pathways and modeling CDG pathophysiology.

COG7 deficiency in Drosophila generates multifaceted developmental, behavioral and protein glycosylation phenotypes.

Congenital disorders of glycosylation (CDG) comprise a family of human multisystemic diseases caused by recessive mutations in genes required for protein N-glycosylation. More than 100 distinct forms of CDGs have been identified and most of them cause severe neurological impairment. The Conserved Oligomeric Golgi (COG) complex mediates tethering of vesicles carrying glycosylation enzymes across the Golgi cisternae. Mutations affecting human COG1, COG2 and COG4-COG8 cause monogenic forms of inherited, autosomal recessive CDGs. We have generated a Drosophila COG7-CDG model that closely parallels the pathological characteristics of COG7-CDG patients, including pronounced neuromotor defects associated with altered N-glycome profiles. Consistent with these alterations, larval neuromuscular junctions of Cog7 mutants exhibit a significant reduction in bouton numbers. We demonstrate that the COG complex cooperates with Rab1 and Golgi phosphoprotein 3 to regulate Golgi trafficking and that overexpression of Rab1 can rescue the cytokinesis and locomotor defects associated with loss of Cog7. Our results suggest that the Drosophila COG7-CDG model can be used to test novel potential therapeutic strategies by modulating trafficking pathways.