The transmembrane and cytosolic domains of equine herpesvirus type 1 glycoprotein D determine Golgi retention by regulating vesicle formation.

Accumulating evidence underscores the pivotal role of envelope proteins in viral secondary envelopment. However, the intricate molecular mechanisms governing this phenomenon remain elusive. To shed light on these mechanisms, we investigated a Golgi-retained gD of EHV-1 (gDEHV-1), distinguishing it from its counterparts in Herpes Simplex Virus-1 (HSV-1) and Pseudorabies Virus (PRV). To unravel the specific sequences responsible for the Golgi retention phenotype, we employed a gene truncation and replacement strategy. The results suggested that Golgi retention signals in gDEHV-1 exhibiting a multi-domain character. The extracellular domain of gDEHV-1 was identified as an endoplasmic reticulum (ER)-resident domain, the transmembrane domain and cytoplasmic tail (TM-CT) of gDEHV-1 were integral in facilitating the protein's residence within the Golgi complex. Deletion or replacement of either of these dual domains consistently resulted in the mutant gDEHV-1 being retained in an ER-like structure. Moreover, (TM-CT)EHV-1 demonstrated a preference for binding to endomembranes, inducing the generation of a substantial number of vesicles, potentially originate from the Golgi complex or the ER-Golgi intermediate compartment. In conclusion, our findings provide insights into the intricate molecular mechanisms governing the Golgi retention of gDEHV-1, facilitating the comprehension of the processes underlying viral secondary envelopment.

A quantitative study of the Golgi retention of glycosyltransferases.

How Golgi glycosyltransferases and glycosidases (hereafter glycosyltransferases) localize to the Golgi is still unclear. Here, we first investigated the post-Golgi trafficking of glycosyltransferases. We found that glycosyltransferases can escape the Golgi to the plasma membrane, where they are subsequently endocytosed to the endolysosome. Post-Golgi glycosyltransferases are probably degraded by ectodomain shedding. We discovered that most glycosyltransferases are not retrieved from post-Golgi sites, indicating that retention rather than retrieval is the primary mechanism for their Golgi localization. We therefore used the Golgi residence time to study Golgi retention of glycosyltransferases quantitatively and systematically. Quantitative analysis of chimeras of ST6GAL1 and either transferrin receptor or tumor necrosis factor α revealed the contributions of three regions of ST6GAL1, namely the N-terminal cytosolic tail, the transmembrane domain and the ectodomain, to Golgi retention. We found that each of the three regions is sufficient for Golgi retention in an additive manner. N-terminal cytosolic tail length negatively affects the Golgi retention of ST6GAL1, similar to effects observed for the transmembrane domain. Therefore, the long N-terminal cytosolic tail and transmembrane domain could act as Golgi export signals for transmembrane secretory cargos. This article has an associated First Person interview with the first author of the paper.

Disease-causing missense mutations within the N-terminal transmembrane domain of GlcNAc-1-phosphotransferase impair endoplasmic reticulum translocation or Golgi retention.

Transport of newly synthesized lysosomal enzymes to the lysosome requires tagging of these enzymes with the mannose 6-phosphate moiety by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), encoded by two genes, GNPTAB and GNPTG. GNPTAB encodes the α and ß subunits, which are initially synthesized as a single precursor that is cleaved by Site-1 protease in the Golgi. Mutations in this gene cause the lysosomal storage disorders mucolipidosis II (MLII) and mucolipidosis III αß (MLIII αß). Two recent studies have reported the first patient mutations within the N-terminal transmembrane domain (TMD) of the α subunit of GlcNAc-1-phosphotransferase that cause either MLII or MLIII αß. Here, we demonstrate that two of the MLII missense mutations, c.80T>A (p.Val27Asp) and c.83T>A (p.Val28Asp), prevent the cotranslational insertion of the nascent GlcNAc-1-phosphotransferase polypeptide chain into the endoplasmic reticulum. The remaining four mutations, one of which is associated with MLII, c.100G>C (p.Ala34Pro), and the other three with MLIII αß, c.70T>G (p.Phe24Val), c.77G>A (p.Gly26Asp), and c.107A>C (p.Glu36Pro), impair retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes. Our results uncover the basis for the disease phenotypes of these patient mutations and establish the N-terminal TMD of GlcNAc-1-phosphotransferase as an important determinant of Golgi localization.

The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

γ-COPI mediates the retention of kAE1 G701D protein in Golgi apparatus - a mechanistic explanation of distal renal tubular acidosis associated with the G701D mutation.

Mutations of the solute carrier family 4 member 1 (SLC4A1) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant.

Intrinsic Oncogenic Function of Intracellular Connexin26 Protein in Head and Neck Squamous Cell Carcinoma Cells.

It has long been known that the gap junction is down-regulated in many tumours. One of the downregulation mechanisms is the translocation of connexin, a gap junction protein, from cell membrane into cytoplasm, nucleus, or Golgi apparatus. Interestingly, as tumours progress and reinforce their malignant phenotype, the amount of aberrantly-localised connexin increases in different malignant tumours including oesophageal squamous cell carcinoma, thus suggesting that such an aberrantly-localised connexin should be oncogenic, although gap junctional connexins are often tumour-suppressive. To define the dual roles of connexin in head and neck squamous cell carcinoma (HNSCC), we introduced the wild-type connexin26 (wtCx26) or the mutant Cx26 (icCx26) gene, the product of which carries the amino acid sequence AKKFF, an endoplasmic reticulum-Golgi retention signal, at the C-terminus and is not sorted to cell membrane, into the human FaDu hypopharyngeal cancer cell line that had severely impaired the expression of connexin during carcinogenesis. wtCx26 protein was trafficked to the cell membrane and formed gap junction, which successfully exerted cell-cell communication. On the other hand, the icCx26 protein was co-localised with a Golgi marker, as revealed by immunofluorescence, and thus was retained on the way to the cell membrane. While the forced expression of wtCx26 suppressed both cell proliferation in vitro and tumorigenicity in mice in vivo, icCx26 significantly enhanced both cell proliferation and tumorigenicity compared with the mock control clones, indicating that an excessive accumulation of connexin protein in intracellular domains should be involved in cancer progression and that restoration of proper subcellular sorting of connexin might be a therapeutic strategy to control HNSCC.

Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop.

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

The regulation of ER export and Golgi retention of ST3Gal5 (GM3/GM4 synthase) and B4GalNAcT1 (GM2/GD2/GA2 synthase) by arginine/lysine-based motif adjacent to the transmembrane domain.

In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention.

A Balance between Transmembrane-Mediated ER/Golgi Retention and Forward Trafficking Signals in Glycophorin-Anion Exchanger-1 Interaction.

Anion exchanger-1 (AE1) is the main erythroid Cl-/HCO3- transporter that supports CO2 transport. Glycophorin A (GPA), a component of the AE1 complexes, facilitates AE1 expression and anion transport, but Glycophorin B (GPB) does not. Here, we dissected the structural components of GPA/GPB involved in glycophorin-AE1 trafficking by comparing them with three GPB variants-GPBhead (lacking the transmembrane domain [TMD]), GPBtail (mainly the TMD), and GP.Mur (glycophorin B-A-B hybrid). GPB-derived GP.Mur bears an O-glycopeptide that encompasses the R18 epitope, which is present in GPA but not GPB. By flow cytometry, AE1 expression in the control erythrocytes increased with the GPA-R18 expression; GYP.Mur+/+ erythrocytes bearing both GP.Mur and GPA expressed more R18 epitopes and more AE1 proteins. In contrast, heterologously expressed GPBtail and GPB were predominantly localized in the Golgi apparatus of HEK-293 cells, whereas GBhead was diffuse throughout the cytosol, suggesting that glycophorin transmembrane encoded an ER/Golgi retention signal. AE1 coexpression could reduce the ER/Golgi retention of GPB, but not of GPBtail or GPBhead. Thus, there are forward-trafficking and transmembrane-driven ER/Golgi retention signals encoded in the glycophorin sequences. How the balance between these opposite trafficking signals could affect glycophorin sorting into AE1 complexes and influence erythroid anion transport remains to be explored.

Retention mechanisms for ER and Golgi membrane proteins.

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins.