The SMS domain of Trs23p is responsible for the in vitro appearance of the TRAPP I complex inSaccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae transport protein particle (TRAPP) is a family of related multisubunit complexes required for endoplasmic reticulum-to-Golgi transport (TRAPP I), endosome-to-Golgi transport (TRAPP II) or cytosol to vacuole targeting (TRAPP III). To gain insight into the relationship between these complexes, we generated random and targeted mutations in the Trs23p core subunit. Remarkably, at physiological salt concentrations only two peaks (TRAPP I and a high molecular weight peak) are detected in wild-type cells. As the salt was raised, the high molecular weight peak resolved into TRAPP II and III peaks. Deletion of a Saccharomycotina-specific domain of Trs23p resulted in destabilization of TRAPP I but had no effect on TRAPP II or III. This mutation had no observable growth phenotype, normal levels of Ypt1p-directed guanine nucleotide exchange factor activity in vivo and did not display any in vivo nor in vitro blocks in membrane traffic. Biochemical analysis indicated that TRAPP I could be produced from the TRAPP II/III peak in vitro by increasing the salt concentration. Our data suggest that the SMS domain of Trs23p is responsible for the in vitro appearance of TRAPP I in S. cerevisiae. The implications of these findings are discussed.

A trs20 Mutation That Mimics an SEDT‐Causing Mutation Blocks Selective and Non‐Selective Autophagy: A Model for TRAPP III Organization

“…When fused to GFP, the protein localizes to the plasma membrane and to small buds in dividing cells (Figure C). Previous studies have shown an accumulation of internal structures in trs85 Δ cells (Figure C). Similarly, we found internal, GFP‐Snc1p‐positive structures in trs20D46Y and trs20ts .…”

“…The subunit composition of TRAPP III differs from TRAPP I by just a single additional protein (Trs85p in TRAPP III), yet the molecular size of TRAPP III is much greater than that of TRAPP I (see Figures and ) and this is not due to oligomerization of Trs85p/TRAPP III (S.B. and M.S., unpublished observation).…”

“…To examine this, we fractionated lysates on a Superose 6 column that we and others previously showed efficiently separates TRAPP I, II and III . The fractions from the column were probed with anti‐Trs23p antibody.…”

Ypt/Rab GTPases and their TRAPP GEFs at the Golgi

“…In fact, its mere existence in vivo has been questioned [57]. However, we argue that because TRAPP complexes tend to form dimers and aggregates [48,58], the low level of TRAPP I in cell lysates might not reflect the in vivo situation. Namely, while TRAPP I might exist in vivo, in cell lysates it associates with the larger TRAPP complexes.…”

TRAPPopathies: An emerging set of disorders linked to variations in the genes encoding transport protein particle (TRAPP)‐associated proteins

“…Subsequent phosphorylation of Sec23 by the Golgi‐localized kinase Hrr25 (casein kinase 1δ in humans) releases TRAPP I from the vesicle allowing fusion to proceed, a process that is conserved in humans . In spite of the elegant work performed on yeast TRAPP I, biochemical studies on the yeast Trs23 protein raised the possibility that TRAPP I is an in vitro artifact and may not exist in living cells, a notion that was recently supported by cell biological studies . This is consistent with the lack of identification of a mammalian TRAPP I equivalent and the inability to express recombinant human TRAPP I, while expression of recombinant yeast TRAPP I has been accomplished .…”