Golgi Traffic and Integrity Depend on N-Myristoyl Transferase-1 in Arabidopsis  

Abstract

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADPribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes.

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis

“…expressed the secreted (SEC)-RFP fusion, a bulk-flow secretion reporter, in hlb1 (Faso et al, 2009;Renna et al, 2013) to determine whether the mutant has defects in constitutive secretion mechanisms. We first examined SEC-RFP in actively elongating root hairs because the tips of these cell types are characterized by active secretion (Preuss et al, 2004), and the most obvious growth and F-actin organizational defects of hlb1 seedlings were observed in root hairs (Figures 3 and 4).…”

“…The PIN2-GFP, Man49-mCherry, SYP61-CFP, mCherry-VTI12, mCherry-RabA1g, mCherry-Ara6, GFP-ABD2-GFP, mCherry-ABD2-mCherry, MIN7/BEN1-GFP, RFP-AFVY, and SEC-RFP lines were described previously (Nelson et al, 2007;Robert et al, 2008;Geldner et al, 2009;Nomura et al, 2011;Feraru et al, 2012;Renna et al, 2013;Dyachok et al, 2014). To generate the UBQ10pro:Lifeact-mEGFP construct, mEGFP with the 51-bp Lifeact sequence directly upstream was amplified from plasmid DNA described by Vidali et al (2009) using the following primers: Lifeact-F-EcoRI (59-CATGAATTCATGGGTGTCGCAGATTTGATC-39) and Lifeact-R-SpeI (59-TACACTAGTTTACTTGTACAGCTCGTCCATGC-39).…”

Advances in Plant ER Architecture and Dynamics

“…In humans, alterations in ER-mediated processes cause disease phenotypes that have been classified into three groups: (1) cargo retention and degradation, (2) cargo accumulation and ER stress, and (3) ER transport machinery diseases (Aridor and Hannan, 2000). Also in plant cells, defects in ER functionality lead to various developmental defects (Tamura et al, 2005;Conger et al, 2011;Stefano et al, 2012;Renna et al, 2013), supporting a critical role of the ER for organism biology at large.…”

Trafficking of the myrosinase‐associated proteinGLL23 requiresNUC/MVP1/GOLD36/ERMO3 and the p24 proteinCYB

“…To test this hypothesis, we made a C‐terminal GLL23–RFP construct and drove expression with the UBQ10 promoter (UBQ10 prom ). We chose mRFP because it is better suited for acidic compartments (Marti et al ., ; Renna et al ., ) than GFP, which displays weak fluorescence under acidic conditions (Tamura et al ., ). In cells of stably transformed lines, GLL23–RFP accumulated in the aberrant structures of nuc (Figure e) and cyb (Figure f and Video S1).…”