Mitotic phosphorylation of rab4 prevents binding to a specific receptor on endosome membranes

Nagi Ayad, Michael Hull, Ira Mellman

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Phosphorylation of the monomeric GTPase rab4 in mitotic cells leads to its relocalization from endosome membranes to the cytosol. To determine the mechanism underlying this change in distribution, we established an in vitro assay that reconstituted specific binding of rab4 when endosome-containing membranes were incubated with rab4 complexed with its cytosolic chaperone, GDP dissociation inhibitor (GDI). rab4 was found to bind to a saturable receptor associated with highly purified endosomes. Membrane binding and nucleotide exchange were physically distinct, since an active soluble fragment of the rab4 receptor, but not rab4 nucleotide exchange activity, could be released from membranes by elastase cleavage. Interestingly, the soluble fragment could be used to fully reconstitute rab4 membrane binding. In vitro phosphorylation of rab4 by cdc2/cyclin B kinase did not affect formation of rab4-GDI complexes, but did completely inhibit rab4 binding to its receptor. In contrast, in vitro phosphorylation of membranes did not result in the dissociation of bound rab4, nor were mitotic membranes deficient with respect to binding non-phosphorylated rab4. Thus, mitotic cells appear to accumulate rab4 in the cytosol by phosphorylating rab4 during the soluble phase of its normal activity cycle, thereby preventing membrane attachment.

Original languageEnglish
Pages (from-to)4497-4507
Number of pages11
JournalEMBO Journal
Volume16
Issue number15
DOIs
StatePublished - Aug 1 1997
Externally publishedYes

Fingerprint

Phosphorylation
Endosomes
Membranes
Guanine Nucleotide Dissociation Inhibitors
Cytosol
Nucleotides
Cyclin B
Activity Cycles
Pancreatic Elastase
GTP Phosphohydrolases
Assays
Phosphotransferases

Keywords

  • Endosomal membrane
  • GTPase
  • Mitosis
  • Phosphorylation
  • rab4

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Mitotic phosphorylation of rab4 prevents binding to a specific receptor on endosome membranes. / Ayad, Nagi; Hull, Michael; Mellman, Ira.

In: EMBO Journal, Vol. 16, No. 15, 01.08.1997, p. 4497-4507.

Research output: Contribution to journalArticle

Ayad, Nagi ; Hull, Michael ; Mellman, Ira. / Mitotic phosphorylation of rab4 prevents binding to a specific receptor on endosome membranes. In: EMBO Journal. 1997 ; Vol. 16, No. 15. pp. 4497-4507.
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