Establishment of the ambient pH signaling complex in Aspergillus nidulans: PalI assists plasma membrane localization of PalH

Department of Microbiology, Imperial College London, Flowers Building, Armstrong Road, London SW7 2AZ, UK; Departmento de Microbiología Molecular, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain; Department of Biology, Eckerd College, 4200 54th Avenue South. St. Petersburg, Florida 33711, USA

^* To whom correspondence should be addressed. Email: penalva{at}cib.csic.es.

	Abstract

The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a 3-TMD containing protein with an N-terminal signal peptide and show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27 kDa form but its absence markedly reduces accumulation of the 53 kDa intermediate after shifting cells to alkaline pH. PalI and their homologues contain a predicted lumenal, conserved Gly-Cys containing motif that distantly resembles a Gly-rich dimerization domain. Gly44Arg and Gly47Asp substitutions within this motif lead to loss-of-function. Gly47Asp prevents plasma membrane localization of PalI-GFP and leads to its missorting into the multivesicular body pathway. Overexpression of the 7-TMD and likely ambient alkaline pH receptor PalH partially suppresses the null palI32 mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, co-expression of PalI to stoichiometrically similar levels results in strong predominance of PalH in the plasma membrane. Thus one, but possibly not the only, role of PalI is assisting the plasma membrane localization of PalH. When considered with previous reports in both Saccharomyces cerevisiae and A. nidulans, these data strongly support the prevailing model of pH signaling, involving two spatially segregated complexes: A plasma membrane complex containing PalH, PalI and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.