The TRAPP Subunit Trs130p Interacts with the GAP Gyp6p to Mediate Ypt6p Dynamics at the Late Golgi

Abstract

Small GTPases of the Rab superfamily participate in virtually all vesicle-mediated trafficking events. Cycling between an active GTP-bound form and an inactive GDP-bound form is accomplished in conjunction with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Rab cascades have been described in which an effector of an activated Rab is a GEF for a downstream Rab, thus ensuring activation of a pathway in an ordered fashion. Much less is known concerning crosstalk between GEFs and GAPs although regulation between these factors could also contribute to the overall physiology of a cell. Here we demonstrate that a subunit of the TRAPP II multisubunit tethering factor, a Rab GEF, participates in the recruitment of Gyp6p, a GAP for the GTPase Ypt6p, to Golgi membranes. The extreme carboxy-terminal portion of the TRAPP II subunit Trs130p is required for the interaction between TRAPP II and Gyp6p. We further demonstrate that TRAPP II mutants, but not a TRAPP III mutant, display a defect in Gyp6p interaction. A consequence of this defective interaction is the enhanced localization of Ypt6p at late Golgi membranes. Although a ypt31/32 mutant also resulted in an enhanced localization of Gyp6p at the late Golgi, the effect was not as dramatic as that seen for TRAPP II mutants, nor was Ypt31/32 detected in the same TRAPP II purification that detected Gyp6p. We propose that the interaction between TRAPP II and Gyp6p represents a parallel mechanism in addition to that mediated by Ypt31/32 for the recruitment of a GAP to the appropriate membrane, and is a novel example of crosstalk between a Rab GAP and GEF.

Bi-allelic mutations in TRAPPC2L result in a neurodevelopmental disorder and have an impact on RAB11 in fibroblasts

“…This, together with the increased levels of active RAB11 observed in fibroblasts from the subjects in the present study, suggest that TRAPPC2L either negatively regulates the GEF activity of TRAPP II towards RAB11 or recruits a GTPase activating protein (GAP) to inactivate RAB11. Both scenarios are consistent with previous findings reporting the weak association (possibly regulatory in nature) of the yeast protein Tca17 with TRAPP II,47 and with the ability of yeast TRAPP II to recruit a GAP protein to the Golgi 48…”

TRAPPC11 functions in autophagy by recruiting ATG2B‐WIPI4/WDR45 to preautophagosomal membranes

“…After the incubation, beads were washed five times with RIPA and four times with low‐detergent buffer (25 mM Tris pH 7.4, 100 mM NaCl, 0.025% SDS). Following the last wash, beads were pelleted and analyzed by mass spectrometry as previously described to identify proteins captured by the BioID purification. Data were analyzed using Scaffold 4 proteomic software.…”

“…To obtain purified TRAPPC11-interacting proteins, BirA-C11 and C11-BirA cell lines were grown in 15 cm dishes. At 60% confluency, they were treated with 50 ng/mL tetracycline for 16 hours at which time the medium was changed and supplemented with 20% FBS, Following the last wash, beads were pelleted and analyzed by mass spectrometry as previously described 77 to identify proteins captured by the BioID purification. Data were analyzed using Scaffold 4 proteomic software.…”

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion

“…25 Based on data from phenomics screens, a new model of Nhx1 function emerged proposing that endocytic defects observed in nhx1∆ cells may be caused by MVB membrane fusion defects (Kallay et al, 2011). Lending the greatest support to this hypothesis is the finding that Nhx1 binds Gyp6 (Ali et al, 2004), a Rab-GTPase Activating Protein (Rab-GAP) that 30 inactivates two Rab-GTPases thought to drive membrane fusion (Strom et al, 1993;Vollmer et al, 1999;Will and Gallwitz, 2001): Ypt6, for MVB -Trans-Golgi Network (TGN) vesicle fusion (Bensen et al, 2001;Luo and Gallwitz, 2003;Suda et al, 2013;Brunet et al, 2016) and Ypt7, for MVB -vacuolar lysosome (or vacuole) fusion (Baldehaar et al, 2010;Epp et al, 2011;Baldehaar et al, 2013;Karim et al, 2017). Knocking out GYP6 partially suppresses protein 35 trafficking defects observed in nhx1∆ cells, suggesting that in the absence of NHX1, Gyp6 may inactivate these Rab-GTPases preventing MVB membrane fusion events (Ali et al, 2004).…”