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1. Chlorovirus PBCV-1 Multidomain Protein A111/114R Has Three Glycosyltransferase Functions Involved in the Synthesis of Atypical N-Glycans

   https://doi.org/10.3390/v13010087  

   Noel, Eric; Notaro, Anna; Speciale, Immacolata; Duncan, Garry A.; De Castro, Cristina; Van Etten, James L. (January 2021, Viruses)

   null (Ed.)

   The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes. 

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2. A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway

   https://doi.org/10.1093/plcell/koae197  

   Liu, Jinling; Chen, Hong; Liu, Li; Meng, Xiangzhao; Liu, Qianwen; Ye, Qinyi; Wen, Jiangqi; Wang, Tao; Dong, Jiangli (July 2024, The Plant Cell)

   Abstract Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here, we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the endoplasmic reticulum (ER) exit sites and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12, and Sec24, and participates in coat protein complex II vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway. 

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3. Critical Determinants in ER-Golgi Trafficking of Enzymes Involved in Glycosylation

   https://doi.org/10.3390/plants11030428  

   Zhang, Ning; Zabotina, Olga A. (February 2022, Plants)

   All living cells generate structurally complex and compositionally diverse spectra of glycans and glycoconjugates, critical for organismal evolution, development, functioning, defense, and survival. Glycosyltransferases (GTs) catalyze the glycosylation reaction between activated sugar and acceptor substrate to synthesize a wide variety of glycans. GTs are distributed among more than 130 gene families and are involved in metabolic processes, signal pathways, cell wall polysaccharide biosynthesis, cell development, and growth. Glycosylation mainly takes place in the endoplasmic reticulum (ER) and Golgi, where GTs and glycosidases involved in this process are distributed to different locations of these compartments and sequentially add or cleave various sugars to synthesize the final products of glycosylation. Therefore, delivery of these enzymes to the proper locations, the glycosylation sites, in the cell is essential and involves numerous secretory pathway components. This review presents the current state of knowledge about the mechanisms of protein trafficking between ER and Golgi. It describes what is known about the primary components of protein sorting machinery and trafficking, which are recognition sites on the proteins that are important for their interaction with the critical components of this machinery. 

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4. Calcineurin B-Like Proteins CBL4 and CBL10 Mediate Two Independent Salt Tolerance Pathways in Arabidopsis

   https://doi.org/10.3390/ijms20102421  

   Yang, Yang; Zhang, Chi; Tang, Ren-Jie; Xu, Hai-Xia; Lan, Wen-Zhi; Zhao, Fugeng; Luan, Sheng (May 2019, International Journal of Molecular Sciences)

   In Arabidopsis, the salt overly sensitive (SOS) pathway, consisting of calcineurin B-like protein 4 (CBL4/SOS3), CBL-interacting protein kinase 24 (CIPK24/SOS2) and SOS1, has been well defined as a crucial mechanism to control cellular ion homoeostasis by extruding Na+ to the extracellular space, thus conferring salt tolerance in plants. CBL10 also plays a critical role in salt tolerance possibly by the activation of Na+ compartmentation into the vacuole. However, the functional relationship of the SOS and CBL10-regulated processes remains unclear. Here, we analyzed the genetic interaction between CBL4 and CBL10 and found that the cbl4 cbl10 double mutant was dramatically more sensitive to salt as compared to the cbl4 and cbl10 single mutants, suggesting that CBL4 and CBL10 each directs a different salt-tolerance pathway. Furthermore, the cbl4 cbl10 and cipk24 cbl10 double mutants were more sensitive than the cipk24 single mutant, suggesting that CBL10 directs a process involving CIPK24 and other partners different from the SOS pathway. Although the cbl4 cbl10, cipk24 cbl10, and sos1 cbl10 double mutants showed comparable salt-sensitive phenotype to sos1 at the whole plant level, they all accumulated much lower Na+ as compared to sos1 under high salt conditions, suggesting that CBL10 regulates additional unknown transport processes that play distinct roles from the SOS1 in Na+ homeostasis. 

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5. Identification of a Golgi-localized peptide reveals a minimal Golgi-targeting motif

   https://doi.org/10.1091/mbc.E22-03-0091  

   Navarro, Alexandra P.; Cheeseman, Iain M. (October 2022, Molecular Biology of the Cell)

   Munson, Mary (Ed.)

   Prior work has identified signal sequences and motifs that are necessary and sufficient to target proteins to specific subcellular regions and organelles such as the plasma membrane, nucleus, endoplasmic reticulum, and mitochondria. In contrast, minimal sequence motifs that are sufficient for Golgi localization remain largely elusive. In this work, we identified a 37–amino acid alternative open reading frame (altORF) within the mRNA of the centromere protein CENP-R. This altORF peptide localizes specifically to the cytoplasmic surface of the Golgi apparatus. Through mutational analysis, we identify a minimal 10–amino acid sequence and a critical cysteine residue that are necessary and sufficient for Golgi localization. Pharmacological perturbations suggest that this peptide undergoes lipid modification to promote its localization. Together, our work defines a minimal sequence that is sufficient for Golgi targeting and provide a valuable Golgi marker for live cell imaging. 

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