## Enzyme Sorting at the Golgi Cisternae  
Several models for Golgi trafficking have been postulated, but the most favored model for membrane traffic within the Golgi is the cisternal maturation model of Golgi transport ([19; 23]; Figure 2). Figure 2: Elaboration of \(N\)-linked Glycans in the Golgi cisternae. \(N\)-glycan processing in the Golgi gives rise to three main classes of glycans: high-mannose, hybrid and complex glycans. Glycoprotein exit the ERGIC and reach the Golgi apparatus with eight or nine mannose residues. The Golgi apparatus is organized into discrete cisternae where the enzymes needed for glycan elaboration are specifically compartmentalized. Mannose trimming enzymes are located in the _cis_ and _medial_-Golgi. The addition of GlcNAc is an essential step to generate hybrid and complex \(N\)-linked glycans in the _trans_-Golgi. Hybrid \(N\)-glycans are generated by the extension of the \(\alpha\)3-arm that received GlcNAc with the addition of Gal, Fuc and SA. Removal of two terminal of the five Man residues, allows branching with an additional GlcNAc residue and synthesis of biantennary complex \(N\)-glycans with Gal, Fuc and SA. The polarized distribution of glycosylation enzymes is maintained through retrograde transport of COPI coated vesicles. A proper cisternal pH (different shades of orange) is needed to control retrograde sorting of enzymes and the association of glycoenzymes with the trafficking cargo. GlcNAc, \(N\)-acetylglucosamine; Man, mannose; Gal, galactose; NeuAc, \(N\)-Acetylneuraminic acid; Fuc, Fucose. According to the cisternal maturation model, as a new _cis_-Golgi cisterna is formed, it is gradually converted by the accumulation of _medial_ and then _trans_ glycosylation enzymes that travel from more mature Golgi compartments in vesicles moving in retrograde direction. Although there is strong support for this model, recent observations of the transport of soluble cargoes in mammalian cells have led researchers to postulate the existence of intercisternal tubules that offer specialized Golgi traffic routes [24]. Retrograde transport and sorting of glycosylation enzymes depends mostly on COPI-coated vesicles, composed of seven different protein subunits \(\alpha\)COP, \(\beta\)COP, \(\gamma\)COP, \(\delta\)COP, \(\varepsilon\)COP and \(\delta\)COP [25]. The COPI coat is recruited from the cytosol to the membrane primarily through binding to activated Arfl GTPase. As coat proteins are recruited, they assemble a cage-like structure that enhances membrane curvature to promote vesicle formation [26; 27]. A recent study showed that COPI carriers can traffic in anterograde as well as retrograde directions, with the small GTPase Cdc42 regulating the bidirectional COPI transport specificity [28]. Golgi glycosylation also requires maintenance of a stringent cisternal pH, which influences retrograde sorting of enzymes and the association of a glycosylation enzyme with a trafficking cargo [29]. Indeed, disruption of the cisternal pH leads to mislocalization of glycosyltransferases [30; 31]. Consistent with the role of the pH in Golgi glycosylation, loss-of-function mutations in _ATP6V0A2_, the gene encoding the membrane-bound V0a2 subunit of the V-ATPase, disrupt retrograde intra-Golgi trafficking, resulting in glycosylation defects [32]. Patients carrying pathogenic variants in _ATP6V0A2_ present with wrinkly skin syndrome, autosomal recessive cuts lax type II and neurological impairment [32]. Recent work showed that Golgi localization of glycosylation enzymes and their function also depend on lipid composition of the Golgi compartments [33; 34]. Disruption of sphingomyelin homeostasis at the _trans_-Golgi network of mammalian cells, by using short-chain ceramide, affects the organization of Golgi membranes, leading to separation of Golgi-resident proteins from each other [33]. As a consequence, the enzyme sialyltransferase is unable to interact with its substrate TGN46, that subsequently fails to be glycosylated.