Subproject A02: pH in the TGN/EE as a specificity determinant for post-Golgi trafficking
Principal investigators:
Prof. Dr. Karin Schumacher
Universität Heidelberg
COS, Entwicklungsbiologie
der Pflanzen
INF 230, 69120 Heidelberg
Tel 06221-54-546436
Fax 06221-54-546404
karin.schumacher@cos.uni-heidelberg.de
Prof. Dr. Ursula Kummer
Universität Heidelberg
COS/Bioquant, Modellierung
biologischer Prozesse
INF 267, 69120 Heidelberg
Tel 06221-54-51278
Fax 06221-54-51483
ursula.kummer@bioquant.uni-heidelberg.de
Summary:
Adaptation of the cellular protein-repertoire according to the developmental program and in response to environmental changes requires a highly specific targeting and trafficking machinery. In plants, the trans-Golgi network/early endosome (TGN/EE) is the central hub in which exo- and endocytic trafficking pathways converge and thus specificity of cargo routing needs to be achieved. The TGN/EE is a highly dynamic compartment and yet a core set of integral membrane proteins appears static in midst of heavy incoming and outgoing traffic and thus specifies TGN/EE identity. Luminal pH is a key determinant for protein trafficking and we have shown that function and maintenance of the TGN/EE are dependent on acidification mediated by V-ATPase complexes containing VHA-a1. Building on the identification of the TGN/EE targeting domain in VHA-a1 we now aim to identify the retention/retrieval mechanism that confines VHA-a1 to the TGN/EE. Overexpression of the N-terminal domain of VHA-a1 causes dominant-negative effects and we will investigate if this is caused by a a pH-sensing function that regulates direct interaction with components of the trafficking machinery. Moreover, we have shown that the precise TGN/EE-localization of the V-ATPase is dependent on the acitivity of two members of the ClC-family of anion transporters pointing to a crucial yet mechanistically unresolved connection between anion homeostasis and protein trafficking. Combining the tools we have established for in vivo pH-measurements with super-resolution microscopy we will not only be able to determine pH in particular subdomains of the TGN/EE but will provide additional input for the mathematical model of the TGN/EE contribution to vacuolar pH. The refined models will allows us to test predictions which players along with the V-ATPases are the determining factors. Moreover, we will establish mathematical models for pH-homeostasis in the TGN/EE that will help us to determine if so far uncharacterised proteins identified in our proteomic and genetic approaches need to be considered.