The Candida albicans Kar2 protein is essential and functions during the translocation of proteins into the endoplasmic reticulum.

Since the secretory pathway is essential for Candida albicans to transition from a commensal organism to a pathogen, an understanding of how this pathway functions may be beneficial for identifying novel drug targets to prevent candidiasis. We have cloned the C. albicans KAR2 gene, which performs many roles during the translocation of proteins into the endoplasmic reticulum (ER) during the first committed step of the secretory pathway in many eukaryotes. Our results show that C. albicans KAR2 is essential, and that the encoded protein rescues a temperature-sensitive growth defect found in a Saccharomyces cerevisiae strain harboring a mutant form of the Kar2 protein. Additionally, S. cerevisiae containing CaKAR2 as the sole copy of this essential gene are viable, and ER microsomes prepared from this strain exhibit wild-type levels of post-translational translocation during in vitro translocation assays. Finally, ER microsomes isolated from a C. albicans strain expressing reduced amounts of KAR2 mRNA are defective for in vitro translocation of a secreted substrate protein, establishing a new method to study ER translocation in this organism. Together, these results suggest that C. albicans Kar2p functions during the translocation of proteins into the ER during the first step of the secretory pathway.

Interactions between EB1 and microtubules: dramatic effect of affinity tags and evidence for cooperative behavior.

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.

Probing interactions between CLIP-170, EB1, and microtubules.

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other +TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of alpha-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both alpha-tubulin and beta-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both alpha-tubulin and beta-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the +TIP network.

Minimal plus-end tracking unit of the cytoplasmic linker protein CLIP-170.

Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (+TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 +TIP behavior is unknown, and many potential models have been presented. Here, by separating the two CLIP-170 CAP-Gly domains and their adjacent serine-rich regions into fragments of varied size, we have characterized the minimal plus-end tracking unit of CLIP-170 in vivo. Each CLIP-170 fragment was also characterized for its tubulin polymerization activity in vitro. We found that the two CAP-Gly domains have different activities, whereas CAP-Gly-1 appears incompetent to mediate either +TIP behavior or MT nucleation, a CLIP-170 fragment consisting of the second CAP-Gly domain and its adjacent serine-rich region can both track MT plus ends in vivo and induce tubulin polymerization in vitro. These observations complement recent work on CLIP-170 fragments, demonstrate that CAP-Gly motifs do not require dimerization for +TIP and polymerization-promoting activities, and provide insight into CLIP-170 function and mechanism.

Substitution of alanine for tyrosine-64 in the fingers subdomain of M-MuLV reverse transcriptase impairs strand displacement synthesis and blocks viral replication in vivo.

A distinctive property of reverse transcriptase is the ability to carry out strand displacement synthesis in the absence of accessory proteins such as helicases or single-strand DNA binding proteins. Structure-function studies indicate that the fingers subdomain in HIV-1 reverse transcriptase contacts the template strand downstream of the primer terminus and is involved in strand displacement synthesis. Based on structural comparisons to the HIV-1 enzyme, we made single amino acid substitutions at the Tyr-64 and Leu-99 positions in the fingers subdomain of the M-MuLV reverse transcriptase to ask whether this subdomain has a similar role in displacement synthesis. In vitro assays comparing non-displacement versus displacement synthesis revealed that substitution of alanine at Tyr-64 generated a reverse transcriptase that was impaired in its capacity to carry out DNA and RNA displacement synthesis without affecting polymerase processivity or RNase H activity. However, substitution of Tyr-64 with phenylalanine and a variety of substitutions at position Leu-99 had no specific effect on displacement synthesis. The Y64A substitution prevented viral replication in vivo, and Y64A virus generated reduced levels of reverse transcription intermediates at all steps beyond the synthesis of minus strong stop DNA. The role of the fingers subdomain and in particular the possible contributions of the Tyr-64 residue in displacement synthesis are discussed.

Requirements for DNA unpairing during displacement synthesis by HIV-1 reverse transcriptase.

DNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO(4) oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (+1 and +2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the +2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the +1 and +2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein.