A factor with a zinc- and phorbol ester-binding domain is necessary for endosome fusion

Alejandro Aballay, Nora G. Arenas, Andrew F.G. Quest, Luis S. Mayorga

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

An inhibitory effect of several zinc chelators on endosome fusion reconstituted in an in vitro system has been recently reported (A. Aballay et al., 1995, Biochem. J. 312, 919-923). The factor that requires zinc for its activity is still unknown. Since the regulatory domain of protein kinase C (PKC) contains cysteine-rich motifs which coordinate zinc, we suspected that PKC or a PKC-like protein might be that factor. To test this hypothesis, we studied the effect of calphostin C, a specific inhibitor of PKC that interacts with the cysteine-rich motif, and PMA (phorbol 12-myristate 13- acetate), an activator of several PKC isoforms that bind to the same region, on endosome fusion. Calphostin C inhibited endosome fusion in a zinc- regulated manner, whereas PMA enhanced endosome fusion. Moreover, fusion was strongly stimulated when both PMA and zinc were added together to zinc- depleted fusion reactions. Inhibitors of the catalytic domain of PKC had no effect on the assay suggesting that the kinase activity is not required. In contrast, a glutathione S-transferase fusion protein containing a cysteine- rich region of the regulatory domain of PKCγ inhibited endosome fusion in a PMA-dependent manner. Western blot analysis demonstrated the presence of proteins containing PKC-like cysteine-rich regions that are released from endosomal fractions by zinc chelators. These results indicate that the previously proposed zinc-dependent factor required for endosome fusion could be either a PKC isoform or a protein containing the phorbol ester-binding domain of PKC.

Original languageEnglish (US)
Pages (from-to)28-34
Number of pages7
JournalExperimental Cell Research
Volume235
Issue number1
DOIs
StatePublished - Aug 25 1997
Externally publishedYes

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Endosomes
Phorbol Esters
Protein Kinase C
Zinc
Cysteine
Acetates
Chelating Agents
Protein Isoforms
Proteins
Zinc Acetate
Nucleic Acid Regulatory Sequences
Glutathione Transferase
Catalytic Domain
Phosphotransferases
Western Blotting
phorbol-12-myristate

ASJC Scopus subject areas

  • Cell Biology

Cite this

A factor with a zinc- and phorbol ester-binding domain is necessary for endosome fusion. / Aballay, Alejandro; Arenas, Nora G.; Quest, Andrew F.G.; Mayorga, Luis S.

In: Experimental Cell Research, Vol. 235, No. 1, 25.08.1997, p. 28-34.

Research output: Contribution to journalArticle

Aballay, Alejandro ; Arenas, Nora G. ; Quest, Andrew F.G. ; Mayorga, Luis S. / A factor with a zinc- and phorbol ester-binding domain is necessary for endosome fusion. In: Experimental Cell Research. 1997 ; Vol. 235, No. 1. pp. 28-34.
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