Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain - PubMed (original) (raw)

Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF) 1 and ARF3 that contains a Sec7-like domain

N Morinaga et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Brefeldin A (BFA) inhibited the exchange of ADP ribosylation factor (ARF)-bound GDP for GTP by a Golgi-associated guanine nucleotide-exchange protein (GEP) [Helms, J.B. & Rothman, J.E. (1992) Nature (London) 360, 352-354; Donaldson, J.G., Finazzi, D. & Klausner, R.D. (1992) Nature (London) 360, 350-352]. Cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, it was no longer BFA-sensitive [Tsai, S.-C., Adamik, R., Moss, J. & Vaughan, M. (1996) Proc. Natl. Acad. Sci. USA 93, 305-309]. We describe here purification from bovine brain cytosol of a BFA-inhibited GEP. After chromatography on DEAE-Sephacel, hydroxylapatite, and Mono Q and precipitation at pH 5.8, GEP was eluted from Superose 6 as a large molecular weight complex at the position of thyroglobulin (approximately 670 kDa). After SDS/PAGE of samples from column fractions, silver-stained protein bands of approximately 190 and 200 kDa correlated with activity. BFA-inhibited GEP activity of the 200-kDa protein was demonstrated following electroelution from the gel and renaturation by dialysis. Four tryptic peptides from the 200-kDa protein had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae (total of 51 amino acids), consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7 that plays a similar role in cellular vesicular transport and Sec7 may be a GEP for one or more yeast ARFs.

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Figures

Figure 1

Figure 1

BFA-inhibited GEP activity as a function of GEP protein. Protein precipitated at pH 5.8 after Mono Q chromatography was dissolved in buffer B containing 1 M NaCl. Protein was determined with the Bio-Rad assay. GEP activity of the indicated amount of protein was assayed without (□) and with (○) 6 μg of BFA. ARF activity without GEP (with or without BFA) was 0.36 nmol/hr. This experiment has been replicated twice.

Figure 2

Figure 2

Chromatography of BFA-sensitive GEP on Superose 6. Protein precipitated at pH 5.8 from pooled active Mono Q fractions was dissolved in buffer B containing 1 M NaCl and applied to a column (1 × 30 cm) of Superose 6, which was eluted with buffer B containing 200 mM NaCl (0.4 ml/min). (A) Samples (20 μl) of column fractions (0.4 ml) were assayed for GEP activity without (•) or with (○) 6 μg of BFA. Protein concentration was quantified as absorbance at 280 nm (□). Positions of elution of thyroglobulin (T, 669 kDa), ferritin (F, 440 kDa), and bovine serum albumin (A, 67 kDa) are indicated by arrowheads. (B) Samples (30 μl) of the indicated fractions were subjected to SDS/PAGE in a 4% gel followed by silver staining. Positions of 190- and 200-kDa proteins are indicated on the right. The first lane contains ≈0.5 μg of myosin (200 kDa). Fraction numbers (No) and GEP activity (nmol/h/20 μl) are indicated below each lane. ARF activity was 1.2 nmol/hr.

Figure 3

Figure 3

Activity of BFA-inhibited GEP eluted from gel after SDS/PAGE. A sample (≈100 units) of pH 5.8 precipitate was treated with SDS sample buffer at room temperature for 30 min before separation of proteins by SDS/PAGE (4% gel). After electrophoresis, the segment of gel containing protein of ≈200 kDa (referring to position of prestained marker myosin) was excised. Protein was electroeluted for 3 hr in buffer containing 0.1% SDS, followed by electrodialysis for 3 hr in the same buffer without SDS and then dialyzed against buffer B. Another sample of ≈100 units (Total) was treated the same way except that electrophoresis was stopped and elution carried out just before proteins entered the separation gel. GEP activities of indicated amounts of the total and 200-kDa protein fractions are shown. The first of each pair of columns is activity without BFA and the second with 6 μg of BFA.

Figure 4

Figure 4

Amino acid sequences of peptides from 200-kDa protein aligned with Sec7 sequences. Protein sequence databases (nonredundant version) were searched with amino acid sequences of nine peptides from the 200-kDa protein using the

blast

algorithm (19) available on the Internet from the National Center for Biotechnology Information (

http://www.ncbi.nlm.nih.gov

). One peptide (pk57) was found to have significant similarity to Sec7. Alignment of sequences of the eight other peptides with Sec7 using

gene works

identified three with significant identity to Sec7. In parentheses is percentage similarity of sequences. ∗, Identical amino acids; †, conservative differences.

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References

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