Comprehensive knockout analysis of the Rab family GTPases in epithelial cells

J Cell Biol. 2019 Jun 3;218(6):2035-2050. doi: 10.1083/jcb.201810134. Epub 2019 May 9.

Abstract

The Rab family of small GTPases comprises the largest number of proteins (∼60 in mammals) among the regulators of intracellular membrane trafficking, but the precise function of many Rabs and the functional redundancy and diversity of Rabs remain largely unknown. Here, we generated a comprehensive collection of knockout (KO) MDCK cells for the entire Rab family. We knocked out closely related paralogs simultaneously (Rab subfamily knockout) to circumvent functional compensation and found that Rab1A/B and Rab5A/B/C are critical for cell survival and/or growth. In addition, we demonstrated that Rab6-KO cells lack the basement membrane, likely because of the inability to secrete extracellular matrix components. Further analysis revealed the general requirement of Rab6 for secretion of soluble cargos. Transport of transmembrane cargos to the plasma membrane was also significantly delayed in Rab6-KO cells, but the phenotype was relatively mild. Our Rab-KO collection, which shares the same background, would be a valuable resource for analyzing a variety of membrane trafficking events.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / metabolism*
  • Dogs
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Gene Knockout Techniques / methods
  • Guanosine Triphosphate / metabolism*
  • HEK293 Cells
  • Humans
  • Intracellular Membranes
  • Madin Darby Canine Kidney Cells
  • Multiprotein Complexes / metabolism*
  • Organelles / physiology*
  • Phenotype
  • Protein Transport
  • RNA, Small Interfering / genetics*
  • rab GTP-Binding Proteins / antagonists & inhibitors*
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Multiprotein Complexes
  • RNA, Small Interfering
  • Guanosine Triphosphate
  • rab GTP-Binding Proteins

Associated data

  • RefSeq/NM_007591
  • RefSeq/NM_000527