Related Experiment Video

Updated: May 24, 2026

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
10:02

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Published on: October 23, 2016

An ordered pathway for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma membrane

Antonio Galindo1, Ana María Calcagno-Pizarelli, Herbert N Arst

  • 1Department of Molecular Medicine, Centro de Investigaciones Biológicas CSIC, Madrid, Spain.

Journal of Cell Science
|February 21, 2012
PubMed

Insights

The fungal pal/RIM pathway, crucial for pH signaling, assembles at plasma membrane cortical structures, not endosomes. This pathway involves Pal proteins and ESCRT machinery, with PalF ubiquitylation triggering assembly at alkaline pH.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The fungal pal/RIM signaling pathway regulates gene expression in response to environmental pH.
  • This pathway involves dedicated proteins and components of ESCRT (Endosomal Sorting Complexes Required for Transport) complexes.
  • Previous studies suggested pH signaling proteins assemble on endosomal platforms.

Purpose of the Study:

  • To investigate the subcellular localization of pH signaling proteins in Aspergillus nidulans.
  • To determine where the pal/RIM pathway components assemble upon activation by alkaline pH.
  • To elucidate the roles of specific ESCRT proteins and Pal proteins in this process.

Main Methods:

  • Subcellular localization studies using fluorescence microscopy (epifluorescence microscopy).
  • Analysis of protein interactions using pull-down assays and co-immunoprecipitation.
  • Investigating the role of specific genes (e.g., vps27Δ, vps4 deficient) in protein complex stability and localization.

Main Results:

  • PalA and PalC, Vps32 interactors, colocalize at alkaline-pH-induced cortical structures, not endosomes.
  • These cortical structures are dependent on PalH, Vps23, Vps32, and PalF, but independent of Vps27.
  • Vps23 interacts with ubiquitylated PalF, and Vps23-GFP is recruited to cortical structures in a PalF- and alkaline pH-dependent manner.
  • PalC and PalA exhibit sequential recruitment to these structures, establishing their order in the pathway.

Conclusions:

  • The fungal pal/RIM pH signaling pathway assembles at plasma membrane-associated cortical structures, challenging the endosomal platform model.
  • The ESCRT machinery, particularly Vps23 and ESCRT-III disassembly, plays a critical role in regulating the dynamics of these signaling complexes.
  • The findings provide a refined model for pH-mediated gene regulation, highlighting a novel plasma membrane-based signaling platform.

More Related Videos

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking11:33

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Published on: December 17, 2013

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System08:54

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Published on: March 20, 2016

Related Concept Videos

Assembly of Signaling Complexes01:30

Assembly of Signaling Complexes

Multiprotein signaling complexes are formed in a dynamic process involving protein-protein interactions at the cytoplasmic domain of transmembrane receptors or enzymatic and non-enzymatic proteins associated with the receptor. These complexes ensure the activation and propagation of intracellular signals that regulate cell functions.
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
Tail-anchoring of Proteins in the ER Membrane01:45

Tail-anchoring of Proteins in the ER Membrane

Tail-anchored, or TA, proteins are estimated to make up to 3-5% of membrane proteins found in the eukaryotic cell. Such proteins have a single transmembrane domain located approximately 30 amino acid residues upstream from the C-terminal end. As a result, the signal recognition particle (SRP) cannot guide a TA protein to the ER membrane for cotranslational insertion. Hence, they are integrated into the ER membrane post-translationally using their C-terminal end as the anchor. TA proteins...
Protein Translocation Machinery on the ER Membrane01:28

Protein Translocation Machinery on the ER Membrane

The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides into the ER lumen and their insertion into the ER membrane.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and produces two-second...
Assembly of the Lipid Bilayer in the ER01:28

Assembly of the Lipid Bilayer in the ER

Biological membranes are more than just a barrier separating cell cytoplasm from the outside environment. They are highly dynamic and help maintain the integrity and physiological stability of the cells as well as membrane-bound organelles. Membranes also play vital roles in cell-to-cell and intracellular communication.
A large chunk of any biological membrane is composed of phospholipids. These lipids have a heterogeneous distribution across different subcellular organelles and even between...
Yeast Signaling01:28

Yeast Signaling

Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...