Related Experiment Video

Updated: May 14, 2026

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry
05:58

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Published on: July 17, 2019

Vac14 protein multimerization is a prerequisite step for Fab1 protein complex assembly and function

Tamadher A Alghamdi1, Cheuk Y Ho, Amra Mrakovic

  • 1Department of Chemistry and Biology and the Molecular Science Program, Ryerson University, Toronto, Ontario, Canada M5B 2K3.

The Journal of Biological Chemistry
|February 8, 2013
PubMed

Insights

Vac14 multimerization is essential for forming the Fab1 complex, which regulates phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) levels critical for endolysosome function and preventing neurodegeneration.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is vital for endolysosome functions, and its dysregulation is linked to neurodegenerative diseases.
  • The synthesis and degradation of PtdIns(3,5)P2 are coordinated by the Fab1 complex, involving the kinase Fab1/PIKfyve and phosphatase Fig4/Sac3.
  • Vac14/ArPIKfyve acts as a scaffolding protein essential for Fab1 complex assembly, but its multimerization properties are poorly understood.

Purpose of the Study:

  • To investigate the role of Vac14 multimerization in the assembly of the Fab1 complex.
  • To determine the molecular mechanisms underlying Vac14 self-interaction and its impact on complex formation.
  • To elucidate the functional consequences of impaired Vac14 multimerization on PtdIns(3,5)P2 homeostasis and cellular processes.

Main Methods:

  • Identification of conserved C-terminal motifs in Vac14 required for self-interaction.
  • Analysis of Vac14 self-interaction using biochemical assays and mutant characterization.
  • Assessment of Fab1 complex assembly and PtdIns(3,5)P2 levels in cells expressing Vac14 mutants.
  • Microscopy to observe vacuole morphology and response to hyperosmotic shock.

Main Results:

  • Conserved C-terminal motifs in Vac14 mediate self-interaction, suggesting Vac14 forms dimers.
  • Monomeric Vac14 mutants fail to interact with Fab1 and Fig4, indicating multimerization is an early step in Fab1 complex assembly.
  • Cells expressing monomeric Vac14 mutants exhibit enlarged vacuoles and impaired vacuole fragmentation upon osmotic stress.
  • These cellular phenotypes correlate with significantly reduced PtdIns(3,5)P2 levels.

Conclusions:

  • Vac14 multimerization into a homocomplex is a prerequisite for Fab1 complex assembly.
  • The Vac14 homocomplex plays a critical role in maintaining cellular PtdIns(3,5)P2 levels.
  • Dysfunctional Vac14 multimerization disrupts endolysosome homeostasis and has implications for neurodegenerative diseases.

More Related Videos

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach09:57

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

Published on: December 17, 2016

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes10:05

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Published on: August 13, 2012

Related Concept Videos

Protein Complex Assembly02:41

Protein Complex Assembly

Proteins can form homomeric complexes with another unit of the same protein or heteromeric complexes with different types.  Most protein complexes self-assemble spontaneously via ordered pathways, while some proteins need assembly factors that guide their proper assembly. Despite the crowded intracellular environment, proteins usually interact with their correct partners and form functional complexes.
Many viruses self-assemble into a fully functional unit using the infected host cell to...
Protein Complex Assembly02:41

Protein Complex Assembly

Proteins can form homomeric complexes with another unit of the same protein or heteromeric complexes with different types.  Most protein complexes self-assemble spontaneously via ordered pathways, while some proteins need assembly factors that guide their proper assembly. Despite the crowded intracellular environment, proteins usually interact with their correct partners and form functional complexes.
Many viruses self-assemble into a fully functional unit using the infected host cell to...
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Mechanism of Filopodia Formation01:39

Mechanism of Filopodia Formation

Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, length, and positioning in different cell types, suggesting their diverse roles.
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...
Generation of Straight or Branched Actin Filaments01:14

Generation of Straight or Branched Actin Filaments

The straight or branched structure formation of actin filaments is controlled by nucleating proteins such as the formins and Arp2/3 complex. Formin-mediated assembly results in straight filaments, whereas Arp2/3 protein complex-mediated assembly results in branched actin filaments.
Arp2/3 Complex
Arp2/3 complex is a seven-subunit complex consisting of two proteins similar to actin- Arp2 and Arp3, and five other subunits that help keep Arp2 and Arp3 inactive. When required, the complex is...