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The Presequence of Euglena LHCPII, a Cytoplasmically Synthesized Chloroplast Protein, Contains a Functional Endoplasmic Reticulum-Targeting Domain

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The Presequence of Euglena LHCPII, a Cytoplasmically Synthesized Chloroplast Protein, Contains a Functional Endoplasmic Reticulum-Targeting Domain University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications in the Biological Sciences Papers in the Biological Sciences 1993 The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain Ram Kishore University of Nebraska - Lincoln Umesh S. Muchhal University of Nebraska - Lincoln Steven D. Schwartzbach University of Nebraska - Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/bioscifacpub Part of the Life Sciences Commons Kishore, Ram; Muchhal, Umesh S.; and Schwartzbach, Steven D., "The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain" (1993). Faculty Publications in the Biological Sciences. 115. https://digitalcommons.unl.edu/bioscifacpub/115 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications in the Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11845-11849, December 1993 Cell Biology The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain (nilcrosomal processing/signal peptide/polyprotein) RAM KISHORE*, UMESH S. MUCHHAL, AND STEVEN D. SCHWARTZBACHt School of Biological Sciences, University of Nebraska, Lincoln, NE 68588 Communicated by Myron K. Brakke, September 20, 1993 (receivedfor review May 5, 1993) ABSTRACT The precursor to the Euglena light-harvest- and 50-70% identity between groups (9). Since GC18 com- ing chlorophyll a/b-binding protein of photosystem II prises slightly more than half of the gene encoding the 6.6-kb (pLHCPII) is unique; it is a polyprotein, synthesized on LHCPII mRNA (9), the complete Euglena pLHCPII poly- membrane-bound ribosomes and transported to the Golgi protein probably contains 8 or 9 LHCPIIs. apparatus prior to chloroplast localization. A cDNA corre- A number of differences have been found between the sponding to the 5' end ofLHCPII mRNA has been isolated and synthesis ofEuglena LHCPII and the synthesis ofLHCPII in sequenced. The deduced amino acid sequence of this cDNA all other organisms. The light-induced synthesis of Euglena indicates that Euglena pLHCPII contains a 141-amino acid pLHCPII is regulated at the level of translational elongation N-terminal extension. The N-terminal extension contains three (11) rather than at transcription. The half-life of Euglena hydrophobic domains and a potential signal peptidase cleavage pLHCPII is 20 min, in contrast to the extremely short halflife site at amino acid 35. Cotranslational processing by canine of pLHCPII in other plants and algae (8, 12). Euglena microsomes removed approximately 35 amino acids from an in pLHCPII is synthesized on membrane-bound ribosomes as vitro synthesized 33-kDa pLHCPII composed of a 141-amino found for proteins transported into the endoplasmic reticu- acid N-terminal extension and a 180-amino acid partial LH- lum (ER) rather than on free ribosomes as normally found for CPU unit truncated at the beginning of the third membrane- cytoplasmically synthesized chloroplast proteins (4, 11). Im- spanning hydrophobic domain. Processed pLHCPII was de- munogold electron microscopy localizes Euglena LHCPII to graded by exogenous protease, indicating that it had not been the Golgi apparatus and chloroplast when LHCPII is being translocated to the microsomal lumen. Extraction with 0.1 M synthesized, while at times when LHCPII is not being Na2CO3, pH 11.5, did not remove the processed pLHCPII from synthesized, an immunoreaction is seen only in the chloro- the microsomal membrane. A stop-transfer membrane anchor plast (10, 13). It thus appears that Euglena pLHCPII is sequence appears to anchor the nascent protein within the transported into the ER prior to chloroplast localization. membrane, preventing translocation into the lumen. Taken In contrast to post-translational transport of proteins into together, these results provide biochemical evidence for a chloroplasts, transport of proteins into the ER is co- functional cleaved signal sequence within the N-terminal ex- translational and dependent upon a 15- to 30-amino acid tension of a Euglena cytoplasmically synthesized chloroplast- N-terminal extension, the signal sequence (3). The signal localized protein. sequence is cleaved on the lumenal side of the ER membrane (3, 14). Signal peptides differ in length and primary sequence The light-harvesting chlorophyll a/b-binding proteins ofpho- (3, 15). All identified signal peptides contain a positively tosystem II (LHCPII) are a group of highly conserved 25- to charged N-terminal region, a hydrophobic core, and a more 28-kDa proteins encoded by a nuclear multigene family polar C-terminal region (3, 15). The signal peptidase cleavage composed of 3-20 members, depending upon the organism site can be predicted on the basis of the amino acid found at studied (1). Differences in gene structure and amino acid the -1 and -3 positions (15, 16). The precursor to a Euglena composition at 14 positions allow LHCPIIs to be classified calcium-binding protein contains a typical eukaryotic three- into three types (2). What if any functional differences exist domain signal peptide at its N terminus (17). This Euglena between LHCPII types remain unclear. precursor is cotranslationally processed by and translocated Chloroplast proteins are synthesized as precursors con- into canine microsomes (17), demonstrating that canine mi- taining an N-terminal extension, the transit peptide, which crosomes can be used as an in vitro system for identifying contains the information required for the posttranslational functional Euglena signal peptides. uptake and proteolytic processing of the precursor by the The precursors to two Euglena cytoplasmically synthe- chloroplast (3, 4). The precursor of LHCPII, pLHCPII, sized chloroplast-localized proteins, the small subunit of typically contains a 29- to 36-amino acid transit peptide (4). ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU) The pLHCPII transit peptide is required for chloroplast and porphobilinogen deaminase (PBGD), contain 134- and localization, while insertion of pLHCPII into the thylakoid 139-amino acid N-terminal extensions (18, 19). The N-termi- membrane appears to utilize targeting signals contained nal extensions do not resemble chloroplast transit peptides, within the mature protein (5-7). Euglena pLHCPIIs are but the N-terminal 35-40 amino acids constitute a signal unique polyproteins encoded by 6.6- and 9.5-kb mRNAs (8). peptide domain with a potential signal peptidase cleavage site A Euglena pLHCPII genomic clone (GC18) encodes five mature LHCPIIs separated by decapeptide linkers (9, 10). Abbreviations: ER, endoplasmic reticulum; Lac, 3-lactamase; The LHCPIIs can be divided into three groups. There is LHCPII, light-harvesting chlorophyll a/b-binding proteins of pho- greater than 90% amino acid sequence identity within a group tosystem II; pLHCPII, precursor to LHCPII (other precursors are named similarly); PBGD, porphobilinogen deaminase; SSU, ribu- lose-1,5-bisphosphate carboxylase/oxygenase small subunit. The publication costs of this article were defrayed in part by page charge *Present address: Department of Cell Biology and Anatomy, New payment. This article must therefore be hereby marked "advertisement" York Medical College, Valhalla, NY 10595. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 11845 11846 Cell Biology: Kishore et al. Proc. Natl. Acad. Sci. USA 90 (1993) + 0000 0 + O o o o o + - LHCP MPNNADAMKFGLAAGAAMGVIVYVLAGAASSTSLA*ATHVNIQQAPAVIPRMASVPSAYTIATNPIGASARVVDAN 75 -+ o -+ 00 00 o o o + o o + 0+ o + ssu MPFDRQPLLSGEKGMPATSLWLVGGAVIAAVC*VIVNTSYNGTQLSVTARPIQAAVSQVSMARFAESGVSRGSGNR + -o -0+ o 0 00 00 + 00 00 +-o PBGD MYCGRYETIGETRGNSLNVFIGAAAGFVAAVA*LINSGLATSFYSTPVRAVPQVIVPSSLAASSQLPVVPKETNIQ -00- 0 0-+00 o +0 + -0 o o oo ++o+ - LHCP YESTDYLTLPATEKSTMGSLLMIAAAGVAAAVAFVWKSVPRQQDSVINVPLLPVSVATMATSGKKSKAAAD 147 0 0 -0-0++ 0 0 + + + 00-+- + ++ - ssu VSQAVPLMAASVGAESESRRWVASAILFPLSGLFAAVALKMA1KPKVAAVLPFTSEKDNVWNPVNNIIIW 0 -oo + -+o o 0 00 + + +00+ - -o + o ooo0 PBGD VNSAQILYPDSTVKGQERTITILGVCSFLSASLFYIWKQFGMKARTTKPADLQEVSGGRIWSLASTTGSNIG FIG. 1. Comparison of the deduced amino acid sequence of the N-terminal extension ofEuglena LHCPII (LHCP), Euglena SSU (18), and Euglena PBGD (19) precursors. Hydrophobic domains (underlined), predicted N-terminal signal peptidase cleavage site (*), hydroxylated amino acids (o), charged amino acids (+, -), and the start of the mature protein (boldface) are indicated. (18-20). The precursor ofPBGD, pPBGD, was not processed pancreatic microsomal membranes (Promega) as described by or transported into canine microsomes, suggesting that the by the supplier. Translation was terminated by the addition signal peptide domain and signal peptidase cleavage site are of 0.1 vol of a solution containing 0.12 M methionine and 0.3 nonfunctional (20). This paper characterizes the N-terminal mM cycloheximide. Protease protection assays were per- extension of Euglena pLHCPII.* As found for pPBGD and formed by incubating the translation mix for 1 h on ice with pSSU, pLHCPII contains a 141-amino acid N-terminal ex- proteinase
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