The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.

Abstract

The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport. Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20. This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene. Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex. Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles. We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

“…Dsl1 forms a complex with Dsl3/Sec39 and Tip20 to form the Dsl1 complex, another member of the CATCHR family of tethering complexes noted for their extended helical rod structures (Lees et al 2010). Further genetic and biochemical dissection of these proteins converged on a role in retrograde transport from the Golgi to the ER: tip20 and dsl1 mutants showed genetic interactions with a variety of ER-Golgi SNAREs (Sweet and Pelham 1993;Andag et al 2001;Kraynack et al 2005), tip20 mutants showed defects in fusion of COPI vesicles (Kamena and Spang 2004), the Dsl1 complex was localized to the ER (Kraynack et al 2005), and Dsl1 interacts directly with multiple components of the COPI coat (Andag and Schmitt 2003).…”

Dsl1p, Tip20p, and the Novel Dsl3(Sec39) Protein Are Required for the Stability of the Q/t-SNARE Complex at the Endoplasmic Reticulum in Yeast

“…Figure 6 shows that a similar set of proteins was isolated regardless of what subunit carried the affinity tag. Six proteins corresponding to Dsl1p (89 k Da), Dsl3p (82 (Sweet and Pelham, 1993) as well as Dsl3p. The TAP-tagged mutant protein was expressed from the DSL1 locus, which was modified by the insertion of plasmid pRS305-⌬Ndsl1 into strain YSC1178 -7502517, thus creating strain D1-⌬C⌬N-T, as described in Materials and Methods.…”

“…In fact, Sec20-TAP-expressing strains carrying the dsl1-22, dsl3-1, tip20-5, and dsl1-⌬N were obtained and analyzed (Supplementary Figure S6B). To facilitate the detection of Sec20p, a series of mutant strains was created that express a c-myc tagged Sec20p version (Sweet and Pelham, 1993).…”

“…Two proteins have been described that may act upstream of, or in conjunction with, the retrograde ER-localized SNAREs. Tip20p, which is an essential ϳ81-kDa peripheral membrane protein that interacts with the cytosolic domain of Sec20p and is involved in Golgi-ER transport (Sweet and Pelham, 1993;Cosson et al, 1997). The second protein is Dsl1p, which was found to be essential for retrograde traffic from the Golgi to the ER.…”

“…In fact, Sec20-TAP-expressing strains carrying the dsl1-22, dsl3-1, tip20-5, and dsl1-⌬N were obtained and analyzed (Supplementary Figure S6B). To facilitate the detection of Sec20p, a series of mutant strains was created that express a c-myc tagged Sec20p version (Sweet and Pelham, 1993).Those mutants that carried point mutations or small deletions (dsl1-22, tip20-5, and sec20-1) were shifted to the restrictive temperature of 35°C for 1 h before the preparation of lysates. Mutant strains carrying large deletions (dsl3-1, dsl1-⌬N) were kept at the permissive temperature of 25°C to avoid artifacts of the temperature shift (see below).…”