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Plant Sar1 isoforms with near-identical protein sequences exhibit different localisations and effects on secretion

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Abstract

In plants, differentiation of subdomains of the endoplasmic reticulum (ER) dedicated to protein export, the ER export sites (ERES), is influenced by the type of export-competent membrane cargo to be delivered to the Golgi. This raises a fundamental biological question: is the formation of transport intermediates at the ER for trafficking to the Golgi always regulated in the same manner? To test this, we followed the distribution and activity of two plant Sar1 isoforms. Sar1 is the small GTPase that regulates assembly of COPII (coat protein complex II) on carriers that transport secretory cargo from ER to Golgi. We show that, in contrast to a tobacco Sar1 isoform, the two Arabidopsis Sar1 GTPases were localised at ERES, independently of co-expression of Golgi-destined membrane cargo in tobacco cells. Although both isoforms labelled ERES, one was found to partition with the membrane fraction to a greater extent. The different distribution of fluorescent fusions of the two isoforms was influenced by the nature of an amino acid residue at the C-terminus of the protein, suggesting that the requirements for membrane association of the two GTPases are not equal. Furthermore, functional analyses based on the secretion of the bulk flow marker α-amylase indicated that over-expression of GTP-restricted mutants of the two isoforms caused different levels of ER export inhibition. These novel results indicate a functional heterogeneity among plant Sar1 isoforms.

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Abbreviations

ER:

Endoplasmic reticulum

ERES:

ER export site

COPII:

Coat protein complex II

GEF:

Guanosine nucleotide exchange factor

YFP:

Yellow fluorescent protein

GFP:

Green fluorescent protein

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Acknowledgements

This work was developed with grants awarded to F.B. from the Canada Foundation for Innovation (CFI), the Canada Research Chair (CRC) program, Natural Science and Engineering Research Council of Canada (NSERC) and Department of Energy, Michigan State University. L.A.M. is supported by an NSERC post-doctoral fellowship.

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Authors and Affiliations

  1. Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, Canada, S7N 5E2

    Sally L. Hanton, Laurent Chatre, Loren A. Matheson, Marika Rossi & Federica Brandizzi

  2. Department of Energy, Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824, USA

    Marika Rossi, Michael A. Held & Federica Brandizzi

Authors
  1. Sally L. Hanton
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  2. Laurent Chatre
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  3. Loren A. Matheson
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  4. Marika Rossi
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  5. Michael A. Held
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  6. Federica Brandizzi
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Corresponding author

Correspondence to Federica Brandizzi.

Additional information

Sally L. Hanton and Laurent Chatre have contributed equally to this work.

Electronic supplementary material

11103_2008_9317_MOESM1_ESM.tif

Comparison of the intracellular localisations of AtSARA1a and AtSARA1b in Arabidopsis leaves. A) AtSARA1a-YFP labels the cytosol (arrow) and punctate structures (arrowheads). B) AtSARA1b-YFP appears to be associated with the ER membranes (arrow) as well as labelling punctate structures (arrowheads) and cytosol Lower panels show magnified areas of the images in A and B to emphasise the differences between the cytosolic localisation of AtSARA1a-YFP and the more reticular pattern exhibited by AtSARA1b-YFP. Bars = 5 μm (TIF 792 kb)

11103_2008_9317_MOESM2_ESM.tif

Comparison of the intracellular localisations of AtSARA1a N159T and AtSARA1b T159N A) AtSARA1aN159T-YFP labels the cytosol (arrow) and punctate structures (arrowheads) in the same manner as wild-type AtSARA1a-YFP. B) AtSARA1bT159N-YFP demonstrates a similar localisation to that of AtSARA1b-YFP. The fluorescence is distributed at punctate structures (arrowheads) and in a reticular pattern comparable to ER labelling (arrow), as well as being partially cytosolic. Lower panels show magnified areas of the images in A and B to emphasise the differences between the cytosolic and reticular patterns exhibited by the position 159 mutants. Bars = 5 μm (TIF 797 kb)

11103_2008_9317_MOESM3_ESM.tif

Alignment of the sequences of Arabidopsis and tobacco Sar1 isoforms. Protein sequences of two Arabidopsis and three tobacco Sar1 isoforms are shown; the three tobacco isoforms are labelled P, A and B, representing accession numbers P52885, AAF17254 and BAA13463 respectively. NtSar1P is the isoform used in previous studies [12, 39, 40]. All five sequences have a high level of similarity, emphasised by grey shading of residues that are identical between at least four of the protein sequences. Note that the GNKXD motif (underlined) is absent only in NtSar1P, and the C-terminal residue in all cases except AtSARA1b is lysine. (TIF 669 kb)

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Hanton, S.L., Chatre, L., Matheson, L.A. et al. Plant Sar1 isoforms with near-identical protein sequences exhibit different localisations and effects on secretion. Plant Mol Biol 67, 283–294 (2008). https://doi.org/10.1007/s11103-008-9317-5

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