Cloning, Zn2+ binding, and structural characterization of the guanine nucleotide exchange factor human Mss4

Hongtao Yu, Stuart L. Schreiber

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The Sec4/Ypt1/Rab family of small GTP-binding proteins are involved in the regulation of intracellular vesicular transport. A rat gene, mss4, that encodes a guanine nucleotide exchange factor (GEF) for Sec4 was recently cloned by its ability to rescue defects in protein transport of a yeast temperature-sensitive (ts) mutant, sec4-8. We describe herein the cloning, bacterial expression, and biochemical characterization of human Mss4. As expected, both the cDNA and its encoded amino acid sequences are highly conserved between the human and rat mss4. Soluble and functional Mss4 protein was obtained by expressing the gene as a glutathione-S-transferase fusion protein in Escherichia coli. Subsequent biochemical analysis revealed that Mss4 binds 1 equiv of Zn2+, and zinc is essential for the stability of the protein. Utilizing multidimensional heteronuclear NMR techniques, we assigned most of the 1H, 15N, and 13C resonances of this 14-kDa protein. Its secondary structure was also deduced from slowly exchanging amide protons, characteristic NOEs, and 3JNH-CαH coupling constants. The protein contains a central seven-stranded antiparallel β sheet, flanked by two small β sheets. Many resonances pertaining to a loop region of the molecule cannot be identified, suggesting that it might be involved in local movements. These studies provide the first structural insights into a protein possessing GEF activity.

Original languageEnglish (US)
Pages (from-to)9103-9110
Number of pages8
JournalBiochemistry
Volume34
Issue number28
StatePublished - 1995

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Guanine Nucleotide Exchange Factors
Cloning
Organism Cloning
Proteins
Biomolecular Nuclear Magnetic Resonance
Protein Stability
Escherichia coli Proteins
Protein Transport
Glutathione Transferase
Rats
GTP-Binding Proteins
Amides
Genes
Protons
Zinc
Amino Acid Sequence
Complementary DNA
Yeasts
Temperature
Yeast

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning, Zn2+ binding, and structural characterization of the guanine nucleotide exchange factor human Mss4. / Yu, Hongtao; Schreiber, Stuart L.

In: Biochemistry, Vol. 34, No. 28, 1995, p. 9103-9110.

Research output: Contribution to journalArticle

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