Rol of transmembrane domain geometry in protein localization to membrane sub-domains in vivo

GONZALO BIGLIANI; JAVIER VALDEZ TAUBAS

Salta

Congreso; 55 SAIB; 2019

SAIB

The geometric features of protein transmembrane domains (TMDs), including the length of the TMD and the volume of the amino acids that constitute their exoplasmic halves, can determine traffic and localization of single spanning membrane proteins. Using transmembrane SNARE as models, we previously showed that short TMDs with high-volume exoplasmic halves are retained in the Golgi, while short TMDs with low-volume exoplasmic halves are transported to the Plasma Membrane (PM). Proteins with long TMDs are transported to the PM regardless of the volume of their exoplasmic halves. Increasing the volume of the residues that constitute the exoplasmic hemi-TMD however, results in a polarised distribution at the PM through endocytic cycling. An interesting possibility is that the length and volume of the exoplasmic hemi-TMD may induce clustering and/or segregation of proteins to membrane subdomains and in turn, this would determine traffic and localisation.In the yeast Saccharomyces cerevisiae, coexisting subdomains have been observed at the plasma membrane. Also, stable micrometre-scale membrane domains are formed in the yeast vacuolar membrane in response to nutrient deprivation and other stresses. Here, we use a set of constructs bearing endogenous and chimeric TMDs of different geometry to analyse whether this is sufficient to confer a differential partition in membrane subdomains in vivo. To compare these proteins, which are normally localised to different organelles, it was necessary to express them in the same membrane. To this end, we changed their cytoplasmic (Sso1) domains, for that of the endosomal/vacuolar SNARE Pep12, which has a signal that could override the sorting information present in the TMDs. Long TMDs were effectively localised to the vacuolar membrane as expected. The chimeric proteins with short TMDs however, localise to the vacuole lumen because they are recognised by a quality control mechanism that involves the ubiquitin ligase Tul1 and the Bsd2 protein. Expression of the chimeras in a bsd2∆tul1∆ mutant strain results in localisation of the proteins at the vacuolar membrane. Finally, we induce micro-domains formation in the vacuolar membrane. These domains were labelled by the liquid disordered (Ld) domain markers Vph1 and FM4-64.Preliminary evidence suggests that the volume of the exoplasmic halves in long TMDs do indeed drive to partition to different membrane subdomains in vivo, suggesting that this phenomenon may underlie their different trafficking behaviour in cells..