Protein Co-localization Studies: Issues and Considerations

Knowledge on the subcellular localization of a newly studied protein is essential for understanding its physiological function in plants. Multiple approaches can be used to determine the subcellular localization of a protein, such as transient co-expression of fluorescent protein-tagged fusion construct with known markers or immunogold transmission electron microscopy (TEM) with specific antibodies. Immunogold TEM is the best method as it reveals the localization of the endogenous protein at the ultra-structural level, which depends on the production of a specific antibody. Alternatively, a fluorescent fusion with the target protein can be another good choice for studying its localization using commercially available antibodies against the fluorescent protein, provided that the fusion is functional (e.g., in a genetic complementation experiment to rescue the knockout mutant of the target protein). In comparison, transient co-expression in protoplasts of the fluorescent protein-tagged target protein with known organelle markers is one of the most favorable methods to determine the subcellular localization of a new protein because of its convenient and time-saving properties, especially for large-scale screening (Denecke et al., 2012Denecke J. Aniento F. Frigerio L. Hawes C. Hwang I. Mathur J. Neuhaus J.M. Robinson D.G. Secretory pathway research: the more experimental systems the better.Plant Cell. 2012; 24: 1316-1326Crossref PubMed Scopus (38) Google Scholar). With the functional fluorescent fusion construct, the subcellular localization of the target protein can be determined or discriminated through comparing the degree of overlapping of the two signals, also known as co-localization, between the target protein and the known organelle marker in confocal imaging. Taking SH3P2 (SH3 domain-containing protein2), a BAR-domain-containing protein that binds to phosphatidylinositol 3-phosphate (PI3P) and regulates autophagosome formation in plants (Zhuang et al., 2013Zhuang X. Wang H. Lam S.K. Gao C. Wang X. Cai Y. Jiang L. A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis.Plant Cell. 2013; 25: 4596-4615Crossref PubMed Scopus (162) Google Scholar), as an example, when singly expressed in Arabidopsis protoplasts, SH3P2-YFP shows both diffuse cytoplasmic and punctate patterns (Figure 1B). However, the punctate dots largely co-localize with ATG9-GFP punctae (Figure 1A) (a membrane marker for autophagosome) upon their co-expression (Figure 1C and 1D), thus demonstrating the autophagosomal localization of SH3P2 in plant cells (Zhuang et al., 2013Zhuang X. Wang H. Lam S.K. Gao C. Wang X. Cai Y. Jiang L. A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis.Plant Cell. 2013; 25: 4596-4615Crossref PubMed Scopus (162) Google Scholar). Cares must be taken in such co-localization investigations because a target protein can be recruited by the marker protein when they are co-expressed in the same cell, thus changing its original distribution pattern. For example, when singly expressed in Arabidopsis protoplasts, the autophagy-related protein ATG6-YFP exhibits a mainly cytosolic pattern with only few punctae (Figure 1E and Supplemental Figure 1A) except under autophagic induction conditions such as nitrogen depletion (Supplemental Figure 1B). This is changed into a definite punctate pattern and largely co-localizes with the autophagosomal marker SH3P2-RFP when co-expressed together (Figure 1G and 1H), indicating that the autophagosomal localization of ATG6-YFP is caused by the recruitment of SH3P2. A similar recruitment phenomenon can also be observed for the protein Exo70E2, an exocyst subunit that labels EXPO (exocyst-positive organelle) for the unconventional protein secretion (UPS) pathway in plant cells (Wang et al., 2010Wang J. Ding Y. Wang J. Hillmer S. Miao Y. Lo S.W. Wang X. Robinson D.G. Jiang L. EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells.Plant Cell. 2010; 22: 4009-4030Crossref PubMed Scopus (181) Google Scholar, Ding et al., 2012Ding Y. Wang J. Wang J. Stierhof Y.D. Robinson D.G. Jiang L. Unconventional protein secretion.Trends Plant Sci. 2012; 17: 606-615Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Exo70E2 can positively recruit other exocyst proteins such as Sec6 to EXPO, because the singly expressed cytosolic pattern of RFP-Sec6 (Figure 1I; Ding et al., 2014Ding Y. Wang J. Lai H.C. Chan H.L. Wang X. Cai Y. Tan X. Bao Y. Xia J. Robinson D.G. et al.Exo70E2 is essential for exocyst subunit recruitment and EXPO formation in both plants and animals.Mol. Biol. Cell. 2014; 25: 412-426Crossref PubMed Scopus (59) Google Scholar) is changed into a punctate pattern and co-localizes fully with the EXPO marker Exo70E2-GFP upon co-expression (Figure 1K and 1L; Ding et al., 2014Ding Y. Wang J. Lai H.C. Chan H.L. Wang X. Cai Y. Tan X. Bao Y. Xia J. Robinson D.G. et al.Exo70E2 is essential for exocyst subunit recruitment and EXPO formation in both plants and animals.Mol. Biol. Cell. 2014; 25: 412-426Crossref PubMed Scopus (59) Google Scholar). Such a recruitment function of Exo70E2 is specific for Sec6 and is not caused by overexpression, because Exo70E2-GFP fails to recruit another cytosolic exocyst subunit, Exo70H2, to EXPO upon co-expression under identical conditions (Supplemental Figure 2A–2D). In addition, free YFP cannot be recruited by either Exo70E2 or SH2P2 (Supplemental Figure 2E–2H and 2I–2L), indicating their specificity in protein recruitment. Protein recruitment by Exo70E2 or SH3P2 can be monitored by direct or indirect protein–protein interaction, which can be tested by Förster resonance energy transfer (FRET) assay in living cells. FRET demonstrates that the recruitment of Sec6 by Exo70E2 to EXPO is the result of a direct protein–protein interaction (FRET efficiency of 10 independent cells was about 20%, which is similar to the positive control Cerulean-linker-EYFP; Gao et al., 2014Gao C. Luo M. Zhao Q. Yang R. Cui Y. Zeng Y. Xia J. Jiang L. A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth.Curr. Biol. 2014; 24: 2556-2563Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar; Figure 1O and 1P). However, such a direct protein–protein interaction is not shown for SH3P2 and ATG6 (FRET efficiency is 0; Figure 1M and 1N), indicating that recruitment of ATG6 by SH3P2 is likely achieved via yet-to-be identified linker proteins that could be localized on the same compartment (Zhuang et al., 2013Zhuang X. Wang H. Lam S.K. Gao C. Wang X. Cai Y. Jiang L. A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis.Plant Cell. 2013; 25: 4596-4615Crossref PubMed Scopus (162) Google Scholar). In addition to protein recruitment and protein–protein interaction, organelle fusion can also result in co-localization of two proteins that are originally located in distinct organelles during a specific biological process such as autophagy. Indeed, EXPO and autophagosome were recently shown to be distinct organelles under normal conditions, but EXPO fuses with autophagosomes for degradation upon autophagic induction in plant cells (Lin et al., 2015Lin Y. Ding Y. Wang J. Shen J. Kung C.H. Zhuang X. Cui Y. Yin Z. Xia Y. Lin H. et al.Exocyst-positive organelles and autophagosomes are distinct organelles in plants.Plant Physiol. 2015; 169: 1917-1932PubMed Google Scholar). For example, during the early stage of expression in cells (13 h after transformation), the autophagosome marker YFP-ATG8e shows a strong cytosolic diffusion pattern with a few dots (which represents the very early stage of autophagy with less autophagosomes; Supplemental Figure 1E), and these autophagosomal dots are separate from the EXPO marker Exo70E2-RFP (Figure 1Q; also seen in Figure 12C and 12D in Wang et al., 2010Wang J. Ding Y. Wang J. Hillmer S. Miao Y. Lo S.W. Wang X. Robinson D.G. Jiang L. EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells.Plant Cell. 2010; 22: 4009-4030Crossref PubMed Scopus (181) Google Scholar). However, when extending the culturing time to 24 h (which represents the late stage of autophagy; Figure 1R and Supplemental Figure 1F), the number and size of YFP-ATG8e punctae increase, showing a high degree of co-localization with Exo70E2-RFP (Figure 1R). Since the Exo70E2-GFP pattern does not show obvious change at different stages when singly expressed in Arabidopsis protoplasts (Supplemental Figure 1G and 1H), these results indicate that EXPO or Exo70E2 can be gradually recruited to autophagosomes during autophagic induction (Lin et al., 2015Lin Y. Ding Y. Wang J. Shen J. Kung C.H. Zhuang X. Cui Y. Yin Z. Xia Y. Lin H. et al.Exocyst-positive organelles and autophagosomes are distinct organelles in plants.Plant Physiol. 2015; 169: 1917-1932PubMed Google Scholar). Such an autophagic-dependent co-localization is also observed between the EXPO marker Exo70E2-RFP and the autophagosome marker SH3P2-YFP (Figure 1S and 1T; Supplemental Figure 1C and 1D). Taken together, using two proteins (SH3P2 and Exo70E2) with the abilities of lipid binding and protein recruitment as examples, we wish to draw the attention of the plant cell and molecular biology community to a new aspect in transient co-expression studies in protoplasts for subcellular localization. As a consequence, we wish to suggest steps or experiments to be considered for studies of this type: (1) single expression of the target protein and organelle marker to determine their respective pattern or “native” localization as reference; (2) co-expression to determine co-localization or separation of the two proteins; (3) evaluate the patterns of single expression versus co-expression for consistency to avoid mis-localization caused by protein recruitment; (4) perform an interaction assay (e.g., FRET) to understand the nature of the protein–protein interaction; and (5) specific attention should be paid to autophagy-related processes that may result in organelle fusion and thus protein co-localization over time. This work was supported by the Research Grants Council of Hong Kong (CUHK466011, 465112, 466613, CUHK2/CRF/11G, C4011–14R, and AoE/M–05/12), the National Natural Science Foundation of China/the Research Grants Council of Hong Kong (grant no. N_CUHK406/12), the National Natural Science Foundation of China (31270226 and 31470294), and the Chinese Academy of Sciences-Croucher Funding Scheme for Joint Laboratories, and Shenzhen Peacock Project (grant no. KQTD201101).