ROL OF TRANSMEMBRANE DOMAIN GEOMETRY IN PROTEIN LOCALIZATION TO MEMBRANE SUB-DOMAINS IN VIVO

Salta

Congreso; LV Annual Meeting of the Argentine Society of Research in Biochemistry and Molecular Biology. (SAIB) and XIV Pan American Societies of Biochemistry and Molecular Biology Congress.; 2019

The geometric features of protein transmembrane domains (TMDs), including the length of the TMD and the volume of the amino acids thatconstitute their exoplasmic halves, can determine traffic and localization of single spanning membrane proteins. Using transmembrane SNAREsas models, we previously showed that short TMDs with high-volume exoplasmic halves are retained in the Golgi, while short TMDs with lowvolume exoplasmic halves are transported to the Plasma Membrane (PM). Proteins with long TMDs are transported to the PM regardless of thevolume of their exoplasmic halves. Increasing the volume of the residues that constitute the exoplasmic hemi-TMD, however, results in a polarizeddistribution at the PM through endocytic cycling. An interesting possibility is that the length and volume of the exoplasmic hemi-TMD may induceclustering and/or segregation of proteins to membrane subdomains and in turn, this would determine traffic and localization. In the yeastSaccharomyces cerevisiae, coexisting subdomains have been observed at the plasma membrane. Also, stable micrometer-scale membrane domainsare formed in the yeast vacuolar membrane in response to nutrient deprivation and other stresses. Here, we use a set of constructs bearingendogenous and chimeric TMDs of different geometry to analyze whether this is sufficient to confer a differential partition in membranesubdomains in vivo. To compare these proteins, which are normally localized to different organelles, it was necessary to express them in the samemembrane. To this end, we changed their cytoplasmic (Sso1) domains, for that of the endosomal/vacuolar SNARE Pep12, which has a signal thatcould override the sorting information present in the TMDs. Long TMDs were effectively localized to the vacuolar membrane as expected. Thechimeric proteins with short TMDs however, localize to the vacuole lumen because they are recognized by a quality control mechanism thatinvolves the ubiquitin ligase Tul1 and the Bsd2 protein. The expression of the chimeras in a bsd2∆tul1∆ mutant strain results in the localization ofthe proteins at the vacuolar membrane. Finally, we induce micro-domains formation in the vacuolar membrane. These domains were labeled bythe liquid disordered (Ld) domain markers Vph1 and FM4-64. Preliminary evidence indicates that the volume of the exoplasmic halves in longTMDs do indeed drive to partition to different membrane subdomains in vivo, suggesting that this phenomenon may underlie their differenttrafficking behavior in cells.