The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco (Nicotiana tabacum) pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.

Several previously unknown low-abundant lipid droplet proteins were identified in Arabidopsis thaliana seeds and seedlings by quantitative proteomics combined with two cell biological approaches.

Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady-state maintenance and turnover of plant LDs, particularly in non-seed tissues, are relatively unknown. Previously, we showed that the LD-associated proteins (LDAPs) are a family of plant-specific, LD surface-associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two-hybrid library using the Arabidopsis LDAP3 isoform as 'bait' in an effort to identify other novel LD protein constituents. One of the candidate LDAP3-interacting proteins was Arabidopsis At5g16550, which is a plant-specific protein of unknown function that we termed LDIP (LDAP-interacting protein). Using a combination of biochemical and cellular approaches, we show that LDIP targets specifically to the LD surface, contains a discrete amphipathic α-helical targeting sequence, and participates in both homotypic and heterotypic associations with itself and LDAP3, respectively. Analysis of LDIP T-DNA knockdown and knockout mutants showed a decrease in LD abundance and an increase in variability of LD size in leaves, with concomitant increases in total neutral lipid content. Similar phenotypes were observed in plant seeds, which showed enlarged LDs and increases in total amounts of seed oil. Collectively, these data identify LDIP as a new player in LD biology that modulates both LD size and cellular neutral lipid homeostasis in both leaves and seeds.

SEIPIN proteins are localized to endoplasmic reticulum (ER)-lipid droplet (LD) junctions where they mediate the directional formation of LDs into the cytoplasm in eukaryotic cells. Unlike in animal and yeast cells, which have single SEIPIN genes, plants have three distinct SEIPIN isoforms encoded by separate genes. The mechanism of SEIPIN action remains poorly understood, and here we demonstrate that part of the function of two SEIPIN isoforms in Arabidopsis (Arabidopsis thaliana), AtSEIPIN2 and AtSEIPIN3, may depend on their interaction with the vesicle-associated membrane protein (VAMP)-associated protein (VAP) family member AtVAP27-1. VAPs have well-established roles in the formation of membrane contact sites and lipid transfer between the ER and other organelles, and here, we used a combination of biochemical, cell biology, and genetics approaches to show that AtVAP27-1 interacts with the N termini of AtSEIPIN2 and AtSEIPIN3 and likely supports the normal formation of LDs. This insight indicates that the ER membrane tethering machinery in plant cells could play a role with select SEIPIN isoforms in LD biogenesis at the ER, and additional experimental evidence in Saccharomyces cerevisiae supports the possibility that this interaction may be important in other eukaryotic systems.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LDIP (LD-Associated Protein [LDAP]-Interacting Protein) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

The small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase is encoded by a nuclear multigene family (rbcS genes). We have cloned and characterized three rbcS cDNA sequences from Brassica napus. These cDNA clones all appear to encode the same protein, but they differ in their nucleotide sequence, which has been exploited in the construction of clone‐specific oligonucleotides as selective hybridization probes. By using a nonselective RNA probe, the temporal expression of the entire rbcS gene family was analyzed during seedling development of Brassica napus and of Sinapis alba. In both crucifer species, rbcS transcripts show transient peak levels and then decrease, although to a different degree. Only a moderate (twofold) difference in transcript pool sizes is observed in light‐grown versus dark‐grown seedlings at the time of peak levels, while a much higher light/dark ratio is found in late‐stage seedlings. The oligonucleotide probes reveal three subsets of transcripts which differ in their accumulation kinetics and light/dark ratio. Assessment of the spatial distribution by using in situ hybridization indicates that rbcS transcripts are uniformly localized in cross‐sections of cotyledons from either light‐grown or dark‐grown seedlings, whereas they are undetectable in root sections.

Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches.

No abstract