An Arabidopsis VPS45p homolog implicated in protein transport to the vacuole

Abstract

The Sec1p family of proteins is required for vesicle-mediated protein trafficking between various organelles of the endomembrane system. This family includes Vps45p, which is required for transport to the vacuole in yeast (Saccharomyces cerevisiae). We have isolated a cDNA encoding a VPS45 homolog from Arabidopsis thaliana (AtVPS45). The cDNA is able to complement both the temperature-sensitive growth defect and the vacuolar-targeting defect of a yeast vps45 mutant, indicating that the two proteins are functionally related. AtVPS45p is a peripheral membrane protein that associates with microsomal membranes. Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations. These results indicate that AtVPS45p is likely to function in the transport of proteins to the vacuole in plants.

Figure 4

Immunoprecipitation of AtVPS45p. Radiolabeled AtVPS45p was generated by in vitro transcription and translation from the cDNA. The translation product (T) was subjected to immunoprecipitation using AtVPS45p antibodies (I) or preimmune serum (P). Samples were analyzed by SDS-PAGE and fluorography.

Figure 1

Sequence comparison of Vps45p-like proteins. Sequences of Vps45p-like proteins from Arabidopsis (AtVPS45p), human (h-Vps45p; GenBank accession no. U35246), and yeast (Vps45p; GenBank accession no. U07972) were compared using single-letter amino acid abbreviations. The alignment was generated using the MegAlign program (DNASTAR, Inc., Madison, WI). Amino acids identical in two or more sequences are shaded.

Figure 2

Complementation of a yeast vps45Δ mutant using AtVPS45. A, Yeast cultures of the vps45Δ mutant (♦), or mutant transformed with yeast VPS45 (□), yeast VPS33 (▴), AtVPS45 (×), or pVT100-U (✽) were grown at 37°C, the temperature at which vps45Δ displays a growth defect. Samples were taken at the times indicated and the number of cells was counted using a hemocytometer. B, The vps45Δ mutant and transformants as described in A were assayed for CPY activity using the APE overlay test. Red colonies indicate CPY activity; colonies lacking activity remain yellow.

Figure 3

Northern analysis of AtVPS45. Thirty micrograms of total RNA from Arabidopsis leaves, roots, flowers, and inflorescence stems was separated on an agarose/formaldehyde gel and transferred to nylon membrane. The membrane was incubated with a ³²P-labeled antisense RNA probe corresponding to the AtVPS45 cDNA. The estimated size of the hybridizing band is indicated on the right.

Figure 5

Association of AtVPS45p with membranes. A, AtVPS45p associates with microsomal membranes. An Arabidopsis root extract was separated into an 8,000g membrane fraction (P8), a 150,000g membrane fraction (P150), and a soluble fraction (S150). Equal amounts of protein from each fraction were separated by SDS-PAGE, transferred to nitrocellulose, and probed using AtVPS45p antibodies. The positions of molecular mass standards are indicated on the right in kilodaltons. B, AtVPS45p is a peripheral membrane protein. A 150,000g membrane pellet from an Arabidopsis root extract was resuspended in extraction buffer alone, 1 m NaCl, 0.1 m Na₂CO₃, 2 m urea, or 1% Triton X-100. Insoluble material was repelleted, and soluble and pellet fractions were analyzed by SDS-PAGE and immunoblotting using AtVPS45p antibodies.

Figure 6

Suc-density gradient fractionation of AtVPS45p. A postnuclear supernatant of an Arabidopsis root extract was analyzed by fractionation on a Suc-density gradient. A, Fractions were analyzed by SDS-PAGE and immunoblotting. Proteins in each fraction were detected using antibodies against AtVPS45p, AtELP (Ahmed et al., 1997), or AtPEP12p (Conceição et al., 1997). B, Blots from A were analyzed by densitometry to determine the relative amount of AtVPS45p (♦), AtELP (▪), or AtPEP12p (▴) in each fraction. C, The concentration of Suc in each fraction was determined using a refractometer.