SMP-1, a Member of a New Family of Small Myristoylated Proteins in Kinetoplastid Parasites, Is Targeted to the Flagellum Membrane inLeishmania

Abstract

The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.

Evolutionary Relationships and Protein Domain Architecture in an Expanded Calpain Superfamily in Kinetoplastid Parasites

“…A similar analysis in L. major revealed that LmCALP20.2 is upregulated in the promastigote insect stage and LmCALP20.1, coded by the adjacent gene, is upregulated in the subsequent metacyclic insect stage (Saxena et al 2003). The flagellar calpain-like protein LmCALP20.10/SMP-1 is detectable only in promastigote stages of Leishmania containing a welldeveloped flagellum, and not in amastigotes, which contain only a highly truncated flagellum (Tull et al 2004). Third, the presence of acylation motifs in many of the proteins, the internal repetitive structure of some of the proteins, and the presence of a cAMP-kinase anchoring protein (AKAP) interaction motif on two proteins indicate that these proteins are associated with membranes or the cytoskeleton.…”

“…In TbCALP4.1CAP5.5, a protein localized to the microtubule cytoskeleton of the cell body, modification of the protein by the addition of myristate and palmitate has been experimentally verified (Hertz-Fowler et al 2001). Recently, it has also been shown that the domain I-only sequence SMP-1 (identical to LmCALP20.10) is also dually acylated and localized to the flagellar membrane in Leishmania (Tull et al 2004). Ablation of acylation by mutating the gene abolishes exclusive flagellar localization and most of the protein remains in the cytosol.…”

“…However, a few common themes emerge from a number of observations. First, the few calpain-like proteins characterized so far at a cellular level are associated with the cytoskeleton or membranes as indicated by biochemical studies and immunolocalizations (Lafaille et al 1989;Muller et al 1992;Hertz-Fowler et al 2001;Tull et al 2004). Second, a number of calpain-like proteins are differentially expressed during the life cycle of the parasites.…”

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

“…To determine whether acylated flagellar proteins are trafficked to the FM in a KH1-dependent manner, we examined the dually acylated TcFCaBP from T. cruzi, which is targeted correctly to the flagellar membrane in Leishmania (38,39), and SMP1 from L. mexicana (40). In both wild type and ⌬kh1 backgrounds, TcFCaBP::HA 3 was targeted correctly to the flagellum in the majority of cells assayed (wild type: 68%, n ϭ 175; ⌬kh1: 69%, n ϭ 145) (Fig.…”

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

“…However, ergosterol and additional cholesterol scavenged by the TbSPT2 RNAi line and by myriocin-treated cells might provide an environment that can support the DRM association of calflagin in the procyclic membrane. Similarly, the Leishmania flagellar membrane protein SMP-1 remains associated with DRMs after myriocin treatment, but not after a combined myriocin and sterol inhibitor treatment (Tull et al, 2004). However, lack of ergosterol and a greater sensitivity to sphingolipid depletion might not allow the bloodstream forms to properly maintain DRM integrity.…”