Homologous Proteins of the Manganese Transporter PAM71 Are Localized in the Golgi Apparatus and Endoplasmic Reticulum

Abstract

Chloroplast manganese transporter 1 (CMT1) and photosynthesis-affected mutant 71 (PAM71) are two membrane proteins that function sequentially to mediate the passage of manganese across the chloroplast envelope and the thylakoid membrane. CMT1 and PAM71 belong to a small five-member protein family in Arabidopsis thaliana. The other three, photosynthesis-affected mutant 71 like 3 (PML3), PML4 and PML5 are not predicted to reside in chloroplast membranes. In this study, the subcellular localization of PML3:GFP, PML4:GFP and PML5:GFP was determined using transient and stable expression assays. PML3:GFP localizes to the Golgi apparatus, whereas PML4:GFP and PML5:GFP are found in the endoplasmic reticulum. We also examined patterns of PML3, PML4 and PML5 promoter activity. Although the precise expression pattern of each promoter was unique, all three genes were expressed in the leaf vasculature and in roots. Greenhouse grown single mutants pml3, pml4, pml5 and the pml4/pml5 double mutant did not exhibit growth defects, however an inspection of the root growth revealed a difference between pml3 and the other genotypes, including wild-type, in 500 µM manganese growth conditions. Strikingly, overexpression of PML3 resulted in a stunted growth phenotype. Putative functions of PML3, PML4 and PML5 are discussed in light of what is known about PAM71 and CMT1.

From the Uncharacterized Protein Family 0016 to the GDT1 family: Molecular insights into a newly-characterized family of cation secondary transporters

“…Indeed, Ca 2+ is a regulator of chloroplast enzymes, and proper modulation of the Ca 2+ concentration within the chloroplast is essential for signaling during photosynthesis [ 4 ]. On the other hand, A. thaliana additionally possesses two ER-localized (PML4 and PML5) and one Golgi-localized (PML3) UPF0016 members [ 2 ]. The putative role in glycosylation of these UPF0016 members at the plant secretory pathway still remains to be investigated, so are their substrate specificity and their overall role in plant cell physiology.…”

“…This protein family was described in 2014 as gathering membrane proteins found in many eukaryotes, bacteria, and archaea [ 1 ]. Numerous paralogs are mainly found in plants, which possess UPF0016 members in various subcellular compartments, including the Golgi [ 2 ], endoplasmic reticulum (ER) [ 2 ], and chloroplasts [ 3 – 7 ], whereas the non-plant eukaryotic UPF0016 members were all found at the Golgi up to now [ 8 , 9 ]. All UPF0016 members are defined by the presence of one or two copies of the Glu-ϕ-Gly-Asp-(Arg/Lys)-(Ser/Thr) consensus motif (with ϕ being any hydrophobic residue) [ 1 ].…”

Gene Replacement in Arabidopsis Reveals Manganese Transport as an Ancient Feature of Human, Plant and Cyanobacterial UPF0016 Proteins

“…It is worth pointing out here that no leaf abnormalities were detected in any of the lines ( Figure 4A ). In contrast, overexpression of the UPF0016 transporter PML3 in the Golgi apparatus of Arabidopsis was accompanied by stunted plant growth and a curled leaf morphology ( Hoecker et al, 2020 ).…”

“…In the model plant Arabidopsis thaliana , the chloroplast manganese transporter1 (CMT1) and photosynthesis-affected mutant71 (PAM71) proteins were found to transport Mn 2+ across the chloroplast envelope and the thylakoid membrane, respectively ( Schneider et al, 2016 ; Eisenhut et al, 2018 ; Zhang et al, 2018 ). Two additional members of the family are located in the endoplasmic reticulum (ER) compartment ( Hoecker et al, 2020 ) and a third, pam71-like3 (PML3), found in the Golgi membrane, facilitates Mn 2+ import under conditions of Mn deficiency ( Yang et al, 2021 ).…”

“…Besides the high degree of conservation, eukaryotic representatives of the UPF0016 family exhibit additional features, among them high sequence diversity within their N-terminal segments ( Hoecker et al, 2017 ). While the ER-localized proteins lack an N-terminal extension, ScGdt1p, HsTMEM165 and AtPML3 all have an N-terminal signal peptide that directs them to the Golgi membrane ( Foulquier et al, 2012 ; Demaegd et al, 2013 ; Hoecker et al, 2020 ; Yang et al, 2021 ). The chloroplast-targeting peptide (cTP) sequences of Arabidopsis PAM71 and CMT1 direct these proteins to the thylakoid and inner-envelope membranes, respectively ( Schneider et al, 2016 ; Eisenhut et al, 2018 ; Zhang et al, 2018 ).…”