Plant Molecular Biology 39: 1–10, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Cloning and characterization of the nuclear AC115 gene of
, Cristen Rosch and Michael R. Kuchka
Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA (
Present address: Ubon Ratchathani University, Ubon Ratchathani, Thailand
Received 26 March 1998; accepted in revised form 6 July 1998
Key words: Chlamydomonas, chloroplast, D2 protein, nuclear gene, photosystem II
The nuclear ac115 mutant of Chlamydomonas reinhardtii is speciﬁcally blocked in the synthesis of the chloroplast
encoded D2 protein of the photosystem II reaction center at a point after translation initiation. Here, we report the
identiﬁcation of the AC115 gene through complementation rescue of the ac115 mutant strain, using an indexed
cosmid library of Chlamydomonas genomic DNA. AC115 is a small, novel, intronless nuclear gene which encodes
a protein of 113 amino acids. The amino terminal end of the Ac115 protein is rich in basic amino acids and has
features which resemble a chloroplast transit sequence. A hydrophobic stretch of amino acids at the protein’s
carboxyl terminus is sufﬁciently large to be a membrane spanning or a protein/protein interaction domain. Various
models are discussed to account for the mechanism by which Ac115p works in D2 synthesis. The ac115 mutant
allele was sequenced and determined to be an A-to-T transversion at the ﬁrst position of the fourth codon of the
coding sequence. This mutation changes an AAG codon to a TAG nonsense codon and results in a null phenotype.
The biogenesis of the chloroplast requires products of
genes of the organellarchromosome which are synthe-
sized within the organelle, together with products of
genes encoded by the nucleus, synthesized in the cyto-
plasm, and transported to their correct location within
the organelle (see for review ). The chloroplast
genome encodes about 100 of its own proteins. The
synthesis of these gene products requires the products
of nuclear genes, some of which act generally at all
major stages of gene expression, others of which be-
have in a gene-speciﬁc fashion (see for review ).
This latter class functions with a surprisingly high de-
gree of speciﬁcity. A single chloroplast gene product
may require the products of many nuclear genes for
its expression. An extreme example is the chloroplast
encoded photosystem I protein gene psaA of Chlamy-
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers AF045466 (AC115 genomic sequence) and
AF045467 (AC115 cDNA sequence).
domonas reinhardtii. The processing of this transcript
is unusually complex in that the mature mRNA is built
of three exons which are spliced together in trans .
This splicing requires the products of a total of at least
14 nuclear genes and one chloroplast gene [11, 12].
We are interested in the expression of the chloro-
plast gene psbD which encodes the photosystem II
(PSII) reaction center protein D2 . The D2 pro-
tein of Chlamydomonas is a trans-thylakoid mem-
brane protein which has ﬁve membrane-spanning α
helices . Speciﬁc amino acids of the D2 protein
have been shown to bind molecules essential for PS II
reaction center function including chlorophyll, pheo-
phytin, plastoquinone A, and a non-heme iron .
D2, together with a related protein D1, the apoproteins
of cytochrome b
, and several associated cofactors,
are responsible for the initial photosynthetic electron
transfer reactions and the generation of a charge sepa-
ration across the thylakoid membrane . Photosys-
tem II is also comprised of a light-harvesting complex,
an oxygen-evolving complex which provides a source
of electrons from water, reaction center ‘core antenna’