The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Cisternae of the Golgi apparatus adhere to each other to form stacks, which are aligned side by side to form the Golgi ribbon. Two proteins, GRASP65 and GRASP55, previously implicated in stacking of cisternae, are shown to be required for the formation of the Golgi ribbon.

Egg yolk phosvitins, generated through the fragmentation of vitellogenins (VTGs), are among the most heavily phosphorylated proteins ever described. Despite the early discovery in 1900 that chicken phosvitin is a phosphoprotein and its subsequent employment as an artificial substrate for a number of protein kinases, the identity of the enzyme(s) responsible for its phosphorylation remained a matter of conjecture until present. Here, we provide evidence that phosvitin phosphorylation is catalyzed by a family with sequence similarity 20, member C (Fam20C), an atypical protein kinase recently identified as the genuine casein kinase and responsible for the phosphorylation of many other secreted proteins at residues specified by the S-x-E/pS consensus. Such a conclusion is grounded on the following observations: (a) the levels of Fam20C and phosphorylated VTG rise in parallel upon treatment of zebrafish with oestrogens; (b) zebrafish phosvitin is readily phosphorylated upon coexpression in U2OS cells with Fam20C, but not with its catalytically inactive mutant; (c) a peptide reproducing a stretch of 12 serines, which are phosphorylated in chicken phosvitin despite lacking the C-terminal priming motif S-x-E, is efficiently phosphorylated by both recombinant and native Fam20C. The last finding expands the repertoire of potential targets of Fam20C to include several proteins known to harbor (p-Ser)n clusters not specified by any known kinase consensus.

The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated 17 mutations in the yeast Golgi-localized Ca2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca2+ -ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation.

The exocyst complex is a multi-subunits evolutionary conserved complex, which was originally shown to be primarily associated with vesicular transport to the plasma membrane. A recent report (Kulich et al., 2013 Traffic; In Press) revealed that AtEXO70B1, one of the multiple subunits of the exocyst complex of Arabidopsis thaliana plants, is co-transported with the autophagy-associated Atg8f protein to the vacuole. This pathway does not involve the Golgi apparatus. The co-localization of AtEXO70B1 and Atg8f suggests either that both of these proteins are co-transported together to the vacuole or, alternatively, that Atg8 binds to a putative Atg8 interacting motif (AIM) located within the AtEXO70B1 polypeptide, apparently forming a tethering complex for an autophagic complex that is transported to the vacuole. In the present addendum, by tooling a bioinformatics approach, we show that AtEXO70B1 as well as the additional 20 paralogs of Arabidopsis EXO70 exocyst subunits each possess one or more AIMs whose consensus sequence implies their high fidelity binding to Atg8. This indicates that the autophagy machinery is strongly involved in the assembly, transport, and apparently also the function of AtEXO70B1 as well as the exocyst sub complex.

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Biogenesis of the regulated secretory pathway in the pancreatic beta-cell involves packaging of products, notably proinsulin, into immature secretory granules derived from the trans-Golgi network. Proinsulin is converted to insulin and C-peptide as granules mature. Secretory proteins not entering granules are conveyed by transport intermediates directly to the plasma membrane for constitutive secretion. One of the co-authors, Peter Arvan, has proposed that in addition, small vesicles bud from granules to traffic to the endosomal system. From there, some proteins are secreted by a (post-granular) constitutive-like pathway. He argues that retention in granules is facilitated by condensation, rendering soluble products (notably C-peptide and proinsulin) more available for constitutive-like secretion. Thus he argues that prohormone conversion is potentially important in secretory granule biogenesis. The other co-author, Philippe Halban, argues that the post-granular secretory pathway is not of physiological relevance in primary beta-cells, and contests the importance of proinsulin conversion for retention in granules. Both, however, agree that trafficking from granules to endosomes is important, purging granules of unwanted newly synthesized proteins and allowing their traffic to other destinations. In this Traffic Interchange, the two co-authors attempt to reconcile their differences, leading to a common vision of proinsulin trafficking in primary and transformed cells.

Previous studies have shown that the Golgi apparatus casein kinase (G-CK) recognizes phosphoacceptor sites specified by the triplet SXE/Sp, which is found in several phosphoproteins, besides casein itself. In the present study, we report that G-CK can phosphorylate, with comparable efficiency, sequences surrounding Ser-22 of salivary proline-rich protein-1 (PRP1), which do not conform to the SXE/Sp motif. By using a series of peptide substrates derived from the PRP1 Ser-22 site, we also have shown that the optimal consensus sequence recognized by G-CK in this case was SXQXX(D/E)3, where the acidic residues at positions n+5 to n+7 and, to a lesser extent, the glutamine residue at position n+2 are the critical determinants.