KETCHing up with Gametogenesis: Nucleocytoplasmic Import and Cell Cycle [OPEN]

In eukaryotes, active import of large signaling molecules and proteins over 40 kD in the nucleus is mediated by aptly named “importin” proteins. As there are considerably fewer importins than there are cargos, determining how cargo-transporter specificity is mediated constitutes a challenging

In eukaryotes, active import of large signaling molecules and proteins over 40 kD in the nucleus is mediated by aptly named "importin" proteins. As there are considerably fewer importins than there are cargos, determining how cargo-transporter specificity is mediated constitutes a challenging issue in the field.
In Arabidopsis (Arabidopsis thaliana), KETCH1 (KARYOPHERIN ENABLING TRANSPORT OF CYTOPLASMIC HYL1) encodes an essential importin responsible for transporting HYL1, a microRNA processor, to the nucleus (Zhang et al., 2017). However, fertility defects resulting from the loss of KETCH1 suggested additional functions. Xiong and colleagues (2020) now report on new functions of KETCH1.
Reexamining the loss-of-function allele ketch1-2/1, they found that seed abortion was close to 50%.
In flowering plants, meiosis produces haploid spores that continue to divide mitotically to produce multicellular organs called gametophytes in which gametes are ultimately formed. The female gametophyte is the embryo sac, and the male gametophyte is the pollen grain. The aborted seeds ratio in ketch1-2/1 plants strongly suggested gametophytic lethality. Indeed, most mutant embryo sacs and pollen grains were unable to develop to maturity and arrested at the first postmeiotic mitotic division. Expression analysis using a KETCH1 promoter to drive a nucleus-localized yellow fluorescent protein (YFP) fusion protein revealed that KETCH1 was expressed throughout embryo sac development and during early stages of pollen grain development.
An attractive hypothesis was that KETCH1 was also responsible for the nuclear import of HYL1 during gametophyte development. Loss of HYL1 results in a low level of sterility (Zhang et al., 2017). However, Xiong and colleagues (2020) did not find evidence of gametophytic sterility in a hyl1 mutant, ruling out that hypothesis.
The team then proceeded to identify other cargos of KETCH1. The high similarity of KETCH1 to mammalian IPO5, which imports ribosomal proteins, suggested that KETCH1 might function similarly. Using bimolecular fluorescence complementation (BiFC) and in vitro pull downs, Xiong and colleagues (2020) found that KETCH1 could interact with ribosomal proteins RPL23A, RPS7, RPS3A, and RPL27a but not with RPL5 and RPL23. Interestingly, RPL27a has been shown to be required for embryo patterning and female gametogenesis (Zsö gö n et al., 2014). Xiong and colleagues (2020) further demonstrate that this interaction is specific, as a closely related importin, IMB4, did not interact with RPL27a.
To validate this interaction in vivo, Xiong and colleagues (2020) introduced GFP-RPL27a and KETCH1-RFP protein fusions driven by the ubiquitous UBQ10 promoter in wild-type and ketch1-2/1 plants. In ketch1-2 pollen and wild-type roots, the accumulation of GFP-RPL27a signal depended on the coexpression of KETCH1-mRFP. GFP-RPL27a also accumulated in response to treatment with the 26S proteasome inhibitor MG132 in roots, suggesting that RPL27a is actively degraded in the absence of KETCH1 (see figure). The ribosomal protein (RP) RPL27a fused to GFP (GFP-RPL27a, green) accumulates only when coexpressed with KETCH1-mRFP (magenta) in pollen grains from ketch1-2/1 plants. Here, Xiong and colleagues (2020) identify a number of additional RPs that require KETCH1 for accumulation. KETCH1-dependent RP stability includes transport to the nucleus and protection from degradation by the 26S proteasome. RP stability conditions ribosome abundance and translation efficiency, which certain genes such as ARF2 and ARF3 are sensitive to, as well as cell cycle progression. (Left panel adapted from Xiong et al. [2020], Figure 8.) [OPEN] Articles can be viewed without a subscription. www.plantcell.org/cgi/doi/10. 1105/tpc.20.00163 To further understand the consequences of the reduced stability of ribosomal proteins, Xiong and colleagues (2020) generated plants in which KETCH1 was constitutively knocked down by a microRNA driven by the 35S promoter (Pro35S::amiR-KETCH1). In these plants, the accumulation of GFP-RLP27a was compromised in leaf protoplasts and pavement cells. Polysome profiling further revealed that these plants had significantly reduced amounts of 40S and 80S ribosomes, consistent with decreased ribosomal protein stability.
Would this reduction in ribosome affect protein translation efficiency? To test this, Xiong and colleagues (2020) generated GFP fusions with the auxin-responsive factors ARF2 and ARF3, which are sensitive to translation efficiency due to an upstream open reading frame in their 59 leader sequence. GFP-ARF2 and GFP-ARF3 signals were reduced in Pro35S:: amiR-KETCH1plants compared with thewild type in an upstream open reading framedependent manner. Reduced translation efficiency can result in cell cycle progression defects in yeast and mammals (Donati et al., 2012), and loss of KETCH1 resulted in mitotic arrest during gametogenesis. The authors further examined Pro35S::amiR-KETCH1 leaves and found that these had fewer and larger pavement cells compared with the wild type. In addition, DNA content profiling revealed increased frequency of pavement cells with 4C and 8C contents compared with the wild type.
Overall, Xiong and colleagues (2020) demonstrate that KETCH1 functions independently of HYL1 in gametophytes, identified ribosomal proteins as cargos of KETCH1, and uncovered a link between translation efficiency and cell cycle progression. These results provide a framework to further understand transporter-cargo specificity in plants.

Sebastien Andreuzza Department of Plant Sciences
University of Cambridge United Kingdom Center for Cellular and Molecular Biology Hyderabad, India sea60@cam.ac.uk ORCID ID: 0000-0002-5547-2692